Inhibition of cataracts and other disorders

ABSTRACT

A method of inhibiting the formation of a cataract in an eye by contacting the eye with a compound having the structure:                    
     is described. Also described is a method of inhibiting the progression of cataract formation in an eye. Methods comprising administering a pharmaceutical composition comprising the above compound to inhibit the formation of a cataract in the eye of a subject and to inhibit progression of cataract formation in the eye of a subject are also described. The above compound also prevents diseases resulting from oxidative stress, including diseases comprising tumor formation resulting from oxidative stress, and also inhibits the progression of diseases resulting from oxidative stress. The above compound may furthermore be used to treat an HIV infection when combined in a pharmaceutical composition with a substance which inhibits HIV replication.

This application is a 321 of PCT/US95/03392 filed Mar. 14, 1995, whichis a CIP of Ser. No. 08/212,569 filed Mar. 14, 1994 now is U.S. Pat. No.5,591,773.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referenced byarabic numerals within parentheses. Full citations for thesepublications may be found at the end of this application, preceding theclaims. The disclosures of these publications in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art to which this invention pertains.Some material incorporated into this application has previously beenpublished by the inventors (76).

Caffeic acid phenethyl ester, hereinafter CAPE, (FIG. 1), an agentoriginally isolated from propolis, a product of honeybee hives, isselectively toxic to transformed cells but not to normal cells (1). Morerecently, CAPE was found to inhibit the transformation mediated byadenovirus type 5 E1nA, as well as the expression of the transformedphenotype in Fischer cloned rat embryo fibroblasts (2). CAPE also wasmore cytotoxic to transformed Fischer cloned rat embryo fibroblasts thanto wild type cells. The growth of other cell lines (NIH 10T{fraction(1/2+L )}, Ltk⁻, and rat 6 cells) transformed with T24 oncogene wasinhibited by CAPE, but not that of the untransformed rat 6 cells (1,2).Propolis has been considered and used in folk medicine as ananti-inflammatory agent with antitumor activity (1). One of the earlieridentified propolis components that possessed anti-inflammatory andbacteriocidal properties was caffeic acid, but CAPE, which is itsphenethyl ester, is more effective. The greater activity of the ester isperhaps due to its ability to more easily pass through the cellmembranes. It is not known whether the free acid or its ester is the onethat is active in vivo.

Reactive oxygen species (ROS), such as superoxide, H₂O₂, and hydroxylradicals, can be generated during aerobic cellular metabolism (3). Inaddition to their significant contribution to mutagenesis,carcinogenesis, and tumor promotion (3, 4), reactive oxygen species havebeen implicated in the etiology and pathophysiology of many humandisease, including rheumatoid arthritis, systemic lupus erythematosus,sickle cell anemia, and various forms of cancer (3, 5, 6). Reactiveoxygen species induce strand breaks in DNA and oxidative modification ofDNA bases, which are implicated in the mutagenic and carcinogeniceffects of reactive oxygen species (3, 7-9). Although the oxidized basescan be repaired by DNA glycosylases and/or endonucleases (10-15), whenthe repair is not complete or timely, deleterious effects may takeplace. 5-hydroxymethyl-2′-deoxyuridine (HMdUrd) is cytotoxic andcytostatic to a number of mammalian cells and is mutagenic, while8-hydroxyl-2′-deoxyguanosine (8-OHdGua) can serve as a mispairing lesionduring cellular DNA replication (9, 15-17). 8-OHdGua has been widelyused as an important biological marker for carcinogenesis and cellularoxidative stress (3, 18, 19).

It has recently been shown that the phorbol ester-type tumor promoters(12-O-tetradecanoylphorbol-13-acetate, hereinafter TPA) induce H₂O₂production in mouse skin as well as cause oxidation of DNA bases in vivo(20-22). In addition, it has been found that agents possessinganti-tumor-promoting properties in vivo, also suppress inflammatoryprocesses. Processes suppressed by such agents include infiltration ofpolymorphonuclear leukocytes (hereinafter PMNs), reactive oxygen speciesproduction, and oxidation of DNA bases (20-22), as well as induction ofornithine decarboxylase (ODC) and edema (23-26). A number of knownanti-tumor promoters that possess all or some of those properties havebeen isolated from biological sources, and include sarcophytol A(isolate from marine soft coral) (27, 28), (−)-epigallocatechin gallate(EGCG, a polyphenol from green tea) (26, 29, 30), curcumin (a spice)(24, 25), and caffeic acid (24, 25).

TPA has been found to induce oxidative stress in bovine eye lens andalso causes its opacity (31). Some of the chemopreventive agents (EGCGand sarcophytol A) used in tumor promotion studies were also effectiveinhibitors of TPA-mediated lens opacification and H₂O₂ production (31).

H₂O₂ and oxygen free radicals participate in cellular aging in humans.Cataract formation in humans is primarily associated with advanced age(32, 33, 34). Indeed, cataract development is one disease thought to betriggered by this kind of oxidative stress (34, 35, 39). In fullydeveloped cataracts in humans and in several experimental cataractmodels, the oxidation of proteins (40, 41) and the peroxidation oflipids in lens tissue appears to be initiated by reactive oxygen species(33, 41-43).

As life expectancy increases, the burden of cataract formation in humansin terms of suffering and cost increases. Worldwide, cataracts are theleading cause of blindness. About 50% of individuals in the UnitedStates over 65 years of age have some stage of cataract development andabout 1.3 million surgical cataract procedures are performed annually(Paton and Craig, 1990). At present, there is no proven non-surgicalmodality to cure cataracts, nor to retard the development of any form ofcataract regardless of age.

Since the mechanism of the action of CAPE is not known, we set out toestablish whether it possesses some of the properties that are common toa number of chemopreventive agents (3). Such properties includeinhibition of ROS production and oxidative damage to cellularmacromolecules, as well as edema and ornithine decarboxylase (ODC)induction, which are thought to contribute to tumor promotion and/orprogression (3, 20-30, 44-47). Furthermore, we decided to establishwhether CAPE prevents TPA-mediated ROS generation by the lens, as wellas lens opacity, properties that would be useful in the prevention ofcataracts (39). We tested the therapeutic use of CAPE to mitigate anddelay the progression of cataracts in intact animals.

The human immunodeficiency virus enzyme HIV integrase mediates theintegration of the HIV DNA into the genome of a host. It has been shownthat CAPE inhibits HIV integrase (48). By combining CAPE with asubstance which inhibits an HIV enzyme other than HIV integrase, HIV maybe blocked at two different replication stages. We disclose herein apharmaceutical composition comprising CAPE and a substance whichinhibits HIV replication, the pharmaceutical composition being usefulfor treating HIV infections.

SUMMARY OF THE INVENTION

This invention provides a method of inhibiting the formation of acataract in an eye, which comprises contacting the eye with an effectivecataract-inhibiting amount of a compound having the structure:

wherein

X and Y are independently carbonyl, C═S, S═O, or O═S═O; n and p areindependently 0 or 1;

R¹ and R² are independently hydrogen, linear or branched C₁₁-C₁₈ alkyl,alkenyl, or alkynyl, either unsubstituted or substituted with halogen,including F, Cl, Br, and I, C₁-C₆ alkoxy, (C═O)R⁷, or (C═O)OR⁸; whereinR⁷ and R⁸ are independently C₁-C₆ linear or branched alkyl;

R³, R⁴, and R⁵ are independently hydrogen, halogen, including F, Cl, Br,and I, trihalomethyl, C₁-C₁₈ linear or branched alkyl, alkenyl, alkynyl,haloalkyl, alkoxy, alkoxyalkyl, alkylthio, or (C═O)R⁹, (C═O)OR¹⁰,O(C═O)R¹¹, (C═S)R¹² (C═S)OR¹³, O(C═S)R¹⁴, (S═O)R¹⁵, (S═O)OR¹⁶, or(O═S═O)OR¹⁷; wherein R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ areindependently C₁-C₁₈ linear or branched alkyl, alkenyl, or alkynyl; and

R⁶ is aryl or C₁-C₁₈ branched or linear alkyl, alkenyl, or alkynyl,either unsubstituted or substituted with halogen, including, F, Cl, Br,and I, C₁-C₆ alkoxy, (C═O)R¹⁸, (C═O)OR¹⁹, or aryl; wherein R¹⁸ and R¹⁹are independently C₁-C₆ branched or linear alkyl;

nor a pharmaceutically acceptable salt thereof.

This invention also provides a method of inhibiting the formation of acataract in an eye of a subject which comprises administering to thesubject a pharmaceutical composition which comprises a pharmaceuticallyacceptable carrier and an effective cataract-inhibiting amount of acompound having the structure:

wherein

X and Y are independently carbonyl, C═S, S═O, or O═S═O; n and p areindependently 0 or 1;

R¹ and R² are independently hydrogen, linear or branched C₁-C₁₈ alkyl,alkenyl, or alkynyl, either unsubstituted or substituted with halogen,including F, Cl, Br, and I, C₁-C₆ alkoxy, (C═O)R⁷, or (C═O)OR⁸; whereinR⁷ and R⁸ are independently C₁-C₆ linear or branched alkyl;

R³, R⁴, and R⁵ are independently hydrogen, halogen, including F, Cl, Br,and I, trihalomethyl, C₁-C₁₈ linear or branched alkyl, alkenyl, alkynyl,haloalkyl, alkoxy, alkoxyalkyl, alkylthio, or (C═O)R⁹, (C═O) OR¹⁰,O(C═O)R¹¹, (C═S)R¹², (C═S)OR¹³, O(C═S)R¹⁴, (S═O)R¹⁵, (S═O)OR¹⁶, or(O═S═O)OR¹⁷; wherein R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ areindependently C₁-C₁₈ linear or branched alkyl, alkenyl, or alkynyl; and

R⁶ is aryl or C₁-C₁₈ branched or linear alkyl, alkenyl, or alkynyl,either unsubstituted or substituted with halogen, including, F, Cl, Br,and I, C₁-C₆ alkoxy, (C═O)R¹⁸, (C═O)OR¹⁹, or aryl; wherein R¹⁸ and R¹⁹are independently C₁-C₆ branched or linear alkyl;

or a pharmaceutically acceptable salt thereof.

This invention also provides a method of inhibiting the progression ofcataract formation in an eye which comprises contacting the eye with aneffective cataract-inhibiting amount of a compound having the structure:

wherein

X and Y are independently carbonyl, C═S, S═O, or O═S═O; n and p areindependently 0 or 1;

R¹ and R² are independently hydrogen, linear or branched C₁-C₁₈ alkyl,alkenyl, or alkynyl, either unsubstituted or substituted with halogen,including F, Cl, Br, and I, C₁-C₆ alkoxy, (C═O)R⁷, or (C═O)OR⁸; whereinR⁷ and R⁸ are independently C₁-C₆ linear or branched alkyl;

R³, R⁴, and R⁵ are independently hydrogen, halogen, including F, Cl, Br,and I, trihalomethyl, C₁-C₁₈ linear or branched alkyl, alkenyl, alkynyl,haloalkyl, alkoxy, alkoxyalkyl, alkylthio, or (C═O)R⁹, (C═O)OR¹⁰,O(C═O)R¹¹, (C═S)R¹², (C═S)OR¹³, O(C═S)R¹⁴, (S═O)R¹⁵, (S═O)OR¹⁶, or(O═S═O)OR¹⁷; wherein R⁹, R¹⁰, R¹¹, R¹², R¹³ R¹⁴, R¹⁵, R¹⁶, and R¹⁷ areindependently C₁-C₁₈ linear or branched alkyl, alkenyl, or alkynyl; and

R⁶ is aryl or C₁-C₁₈ branched or linear alkyl, alkenyl, or alkynyl,either unsubstituted or substituted with halogen, including, F, Cl , Br,and I, C₁-C₆ alkoxy, (C═O)R¹⁸, (C═O)OR¹⁹, or aryl; wherein R¹⁸ and R¹⁹are independently C₁-C₆ branched or linear alkyl;

or a pharmaceutically acceptable salt thereof.

This invention also provides a method of inhibiting the progression ofcataract formation in an eye of a subject which comprises administeringto the subject a pharmaceutical composition which comprises apharmaceutically acceptable carrier and an effective cataract-inhibitingamount of a compound having the structure:

wherein

X and Y are independently carbonyl, C═S, S═O, or O═S═O; n and p areindependently 0 or 1;

R¹ and R² are independently hydrogen, linear or branched C₁-C₁₈ alkyl,alkenyl, or alkynyl, either unsubstituted or substituted with halogen,including F, Cl, Br, and I, C₁-C₆ alkoxy, (C═O)R⁷, or (C═O)OR⁸; whereinR⁷ and R⁸ are independently C₁-C₆ linear or branched alkyl;

R³, R⁴, and R⁵ are independently hydrogen, halogen, including F, Cl, Br,and I, trihalomethyl, C₁-C₁₈ linear or branched alkyl, alkenyl, alkynyl,haloalkyl, alkoxy, alkoxyalkyl, alkylthio, or (C═O)R⁹, (C═O)OR¹⁰,O(C═O)R¹¹, (C═S)R¹², (C═S)OR¹³, O(C═S)R¹⁴, (S═O)R¹⁵, (S═O)OR¹⁶, or(O═S═O)OR¹⁷; wherein R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ areindependently C₁-C₁₈ linear or branched alkyl, alkenyl, or alkynyl; and

R⁶ is aryl or C₁-C₁₈ branched or linear alkyl, alkenyl, or alkynyl,either unsubstituted or substituted with halogen, including, F, Cl, Br,and I, C₁-C₆ alkoxy, (C═O)R¹⁸, (C═O)OR¹⁹, or aryl; wherein R¹⁸ and R¹⁹are independently C₁-C₆ branched or linear alkyl;

or a pharmaceutically acceptable salt thereof.

This invention also provides a method of preventing in a subject adisease resulting from oxidative stress which comprises administering tothe subject a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and an effective oxidative stress-inhibiting amountof a compound having the structure:

wherein

X and Y are independently carbonyl, C═S, S═O, or O═S═O; n and p areindependently 0 or 1;

R¹ and R² are independently hydrogen, linear or branched C₁-C₁₈ alkyl,alkenyl, or alkynyl, either unsubstituted or substituted with halogen,including F, Cl, Br, and I, C₁-C₆ alkoxy, (C═O)R⁷, or (C═O)OR⁸; whereinR⁷ and R⁸ are independently C₁-C₆ linear or branched alkyl;

R³, R⁴, and R⁵ are independently hydrogen, halogen, including F, Cl, Br,and I, trihalomethyl, C₁-C₁₈ linear or branched alkyl, alkenyl, alkynyl,haloalkyl, alkoxy, alkoxyalkyl, alkylthio, or (C═O)R⁹, (C═O)OR¹⁰,O(C═O)R¹¹, (C═S)R¹², (C═S)OR¹³, O(C═S)R₁₄, (S═O)R¹⁵, (S═O)OR¹⁶, or(O═S═O)OR¹⁷; wherein R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ areindependently C₁-C₁₈ linear or branched alkyl, alkenyl, or alkynyl; and

R⁶ is aryl or C₁-C₁₈ branched or linear alkyl, alkenyl, or alkynyl,either unsubstituted or substituted with halogen, including, F, Cl, Br,and I, C₁-C₆ alkoxy, (C═O)R¹⁸, (C═O)OR¹⁹, or aryl; wherein R¹⁸ and R¹⁹are independently C₁-C₆ branched or linear alkyl;

or a pharmaceutically acceptable salt thereof.

This invention also provides a method of inhibiting in a subject theprogression of a disease resulting from oxidative stress which comprisesadministering to the subject a pharmaceutical composition comprising apharmaceutically acceptable carrier and an effective oxidativestress-inhibiting amount of a compound having the structure:

wherein

X and Y are independently carbonyl, C═S, S═O, or O═S═O; n and p areindependently 0 or 1;

R¹ and R² are independently hydrogen, linear or branched C₁-C₁₈ alkyl,alkenyl, or alkynyl, either unsubstituted or substituted with halogen,including F, Cl, Br, and I, C₁-C₆ alkoxy, (C═O)R⁷, or (C═O)OR⁸; whereinR⁷ and R⁸ are independently C₁-C₆ linear or branched alkyl; R³, R⁴, andR⁵ are independently hydrogen, halogen, including F, Cl, Br, and I,trihalomethyl, C₁-C₁₈ linear or branched alkyl, alkenyl, alkynyl,haloalkyl, alkoxy, alkoxyalkyl, alkylthio, or (C═O)R⁹, (C═O)OR¹⁰,O(C═O)R¹¹, (C═S)R¹², (C═S)OR¹³, O(C═S)R¹⁴, (S═O)R¹⁵, (S═O)OR¹⁶, or(O═S═O)OR¹⁷; wherein R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ areindependently C₁-C₁₈ linear or branched alkyl, alkenyl, or alkynyl; and

R⁶ is aryl or C₁-C₁₈ branched or linear alkyl, alkenyl, or alkynyl,either unsubstituted or substituted with halogen, including, F, Cl, Br,and I, C₁-C₆ alkoxy, (C═O)R¹⁸, (C═O)OR¹⁹, or aryl; wherein R¹⁸ and R¹⁹are independently C₁-C₆ branched or linear alkyl;

or a pharmaceutically acceptable salt thereof.

This invention also provides a method of treating a subject having anHIV infection which comprises administering to the subject apharmaceutical composition comprising a pharmaceutically acceptablecarrier, a substance which inhibits HIV replication, and an effectiveHIV integrase-inhibiting amount of a compound having the structure:

wherein

X and Y are independently carbonyl, C═S, S═O, or O═S═O; n and p areindependently 0 or 1;

R¹ and R² are independently hydrogen, linear or branched C₁-C₁₈ alkyl,alkenyl, or alkynyl, either unsubstituted or substituted with halogen,including F, Cl, Br, and I, C₁-C₆ alkoxy, (C═O)R⁷, or (C═O)OR⁸; whereinR⁷ and R⁸ are independently C₁-C₆ linear or branched alkyl;

R³, R⁴, and R⁵ are independently hydrogen, halogen, including F, Cl, Br,and I, trihalomethyl, C₁-C₁₈ linear or branched alkyl, alkenyl, alkynyl,haloalkyl, alkoxy, alkoxyalkyl, alkylthio, or (C═O)R⁹, (C═O)OR¹⁰,O(C═O)R¹¹, (C═S)R¹², (C═S)OR¹³, O(C═S)R¹⁴, (S═O)R¹⁵, (S═O)OR¹⁶, or(O═S═O)OR¹⁷; wherein R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ areindependently C₁-C₁₈ linear or branched alkyl, alkenyl, or alkynyl; and

R⁶ is aryl or C₁-C₁₈ branched or linear alkyl, alkenyl, or alkynyl,either unsubstituted or substituted with halogen, including, F, Cl, Br,and I, C₁-C₆ alkoxy, (C═O)R₁₈, (C═O)OR¹⁹, or aryl; wherein R¹⁸ and R¹⁹are independently C₁-C₆ branched or linear alkyl;

or a pharmaceutically acceptable salt thereof.

Finally, this invention provides a pharmaceutical composition fortreating an HIV infection in a subject which comprises apharmaceutically acceptable carrier, a substance which inhibits HIVreplication, and an effective HIV integrase-inhibiting amount of acompound having the structure:

wherein

X and Y are independently carbonyl, C═S, S═O, or O═S═O; n and p areindependently 0 or 1;

R¹ and R² are independently hydrogen, linear or branched C₁-C₁₈ alkyl,alkenyl, or alkynyl, either unsubstituted or substituted with halogen,including F, Cl, Br, and I, C₁-C₆ alkoxy, (C═O)R⁷, or (C═O)OR⁸; whereinR⁷ and R⁸ are independently C₁-C₆ linear or branched alkyl;

R³, R⁴, and R⁵ are independently hydrogen, halogen, including F, Cl, Br,and I, trihalomethyl, C₁-C₁₈ linear or branched alkyl, alkenyl, alkynyl,haloalkyl, alkoxy, alkoxyalkyl, alkylthio, or (C═O)R⁹, (C═O)OR¹⁰,O(C═O)R¹¹, (C═S)R¹², (C═S)OR¹³, O(C═S)R¹⁴, (S═O)R¹⁵, (S═O)OR¹⁶, or(O═S═O)OR¹⁷; wherein R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ areindependently C₁-C₁₈ linear or branched alkyl, alkenyl, or alkynyl; and

R⁶ is aryl or C₁-C₁₈ branched or linear alkyl, alkenyl, or alkynyl,either unsubstituted or substituted with halogen, including, F, Cl, Br,and I, C₁-C₆ alkoxy, (C═O)R¹⁸, (C═O)OR¹⁹, or aryl; wherein R¹⁸ and R¹⁹are independently C₁-C₆ branched or linear alkyl;

or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Structure of caffeic acid phenethyl ester (CAPE).

FIGS. 2A-2D. CAPE-mediated inhibition of TPA-induced oxidative processesin SENCAR mice. Mice were topically treated with 6.5 nmol CAPE 30 minprior to 6.5 nmol TPA, and this regimen was repeated 20 h later. Micewere sacrificed 1 h after the last treatment and the skin removed fordetermination of: (FIG. 2A) PMN infiltration (by measuringmyeloperoxidase (MPO)); (FIG. 2B) H₂O₂; (FIG. 2C) HMdUrd (HMdU); and(FIG. 2D) 8-OHdGua (8-OHdG), according to procedures described in the“Experimental Details” Section, infra. □, controls treated with acetonein place of CAPE and TPA; □, treatment with acetone followed by TPA; ▪,treatment with CAPE followed by TPA. Results are expressed as mean±SE(bars) from two experiments (2 mice/experiment) in the CAPE series. Datafor acetone (ACT)- and TPA-treated mean values were collected from over20 experiments.

FIG. 3. Effect of CAPE on TPA-induced PMN infiltration and edema inSENCAR mouse ears. These were paired experiments, in which both ears ofeach mouse (8-9/group) were treated with 0.4 nmol TPA±CAPE in 20 μlacetone, and the mice were sacrificed 5 h later. (A) One ear was frozenfor subsequent determination of MPO (a measure of PMN infiltration). (B)A punch (6 mm diameter) was taken from the second ear and weighed tomeasure edematous response (weight of sample minus weight of control).(A) Ears of each group (7-9/group) were separately minced andhomogenized under different conditions as described in the “ExperimentalDetails” Section, infra. MPO was determined in duplicate. MPO wasundetectable in acetone controls and was 4.13±0.0 units/mg protein and1.18±0.1 units/mg protein in the 2 homogenates of TPA-only-treated mice.CAPE-mediated inhibition was calculated versus the appropriate TPA-onlysample, which was homogenized the same way. (B) Weights of ear punches(8-9/group, each determined separately). Acetone controls, 7.2±0.1 mg;TPA in acetone, 16.7±0.7 mg.

FIG. 4. CAPE-mediated inhibition of H₂O₂ formation by TPA-stimulatedhuman PMNs. Cells were incubated with CAPE (0.05-5 nmol/ml) and TPA (25pmol/2.5×10⁵ PMNs/ml) at 37° C. for 30 min, and H₂O₂ was determined bythe phenol red/HRPO assay, as described in the “Experimental Details”Section, infra. Note the semilog scale. The results are presented asmean values±SE.

FIGS. 5A-5C. CAPE-mediated protection of bovine lens from TPA-inducedopacity. Lenses were incubated with artificial aqueous humor only (FIG.5A); incubated with 0.1 μM TPA (FIG. 5B); and pretreated with 1 μM CAPEfor 30 min followed by 0.1 μM TPA (FIG. 5C) for 24 h. After incubation,the lenses were photographed against 3-mm grids.

FIG. 6. The TPA-treated right eye of rabbit No. 5 at the termination ofthe experiment. Specifics relating to the photography are defined in the“Experimental Details” Section, infra.

FIG. 7. The TPA plus CAPE-treated left eye of the same test rabbit(shown in FIG. 6) at the conclusion of the experiment.

FIG. 8. Histogram showing cataract sizes in relative units for all ofthe test rabbits at the termination of the experiment. The solid bars(▪) indicate the left eye treated with TPA and CAPE. □ indicates theright eye, treated with TPA only.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method of inhibiting the formation of acataract in an eye, which comprises contacting the eye with an effectivecataract-inhibiting amount of a compound having the structure:

wherein

X and Y are independently carbonyl, C═S, S═O, or O═S═O; n and p areindependently 0 or 1;

R¹ and R² are independently hydrogen, linear or branched C₁-C₁₈ alkyl,alkenyl, or alkynyl, either unsubstituted or substituted with halogen,including F, Cl, Br, and I, C₁-C₆ alkoxy, (C═O)R⁷, or (C═O)OR⁸; whereinR⁷ and R⁸ are independently C₁-C₆ linear or branched alkyl;

R³, R⁴, and R⁵ are independently hydrogen, halogen, including F, Cl, Br,and I, trihalomethyl, C₁-C₁₈ linear or branched alkyl, alkenyl, alkynyl,haloalkyl, alkoxy, alkoxyalkyl, alkylthio, or (C═O)R⁹, (C═O)OR¹⁰,O(C═O)R¹¹, (C═S)R¹², (C═S)OR¹³, O(C═S)R¹⁴, (S═O)R¹⁵, (S═O)OR¹⁶, or(O═S═O)OR¹⁷; wherein R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ areindependently C₁-C₁₈ linear or branched alkyl, alkenyl, or alkynyl; and

R⁶ is aryl or C₁-C₁₈ branched or linear alkyl, alkenyl, or alkynyl,either unsubstituted or substituted with halogen, including, F, Cl, Br,and I, C₁-C₆ alkoxy, (C═O)R¹⁸, (C═O)OR¹⁹, or aryl; wherein R¹⁸ and R¹⁹are independently C₁-C₆ branched or linear alkyl;

or a pharmaceutically acceptable salt thereof.

As used herein, the phrase “halo” in the terms “haloalkyl” and“trihalomethyl” is intended to mean F, Cl, Br, or I.

Methods of synthesizing caffeic acid esters which may be used tosynthesize compounds having the above-defined structure have beendisclosed in Nakanishi et al., U.S. Pat. No. 5,008,441, the disclosureof which is hereby incorporated by reference. Further examples of theabove-defined compound can be readily synthesized by one of ordinaryskill in the art based on the disclosure of U.S. Pat. No. 5,008,441 andusing techniques generally known to those of ordinary skill. Examples ofsuch methods include the general organic synthesis techniques disclosedin such texts as March, J. Advanced Organic Chemistry, 3rd ed. (Wiley;New York: 1985), the contents of which are hereby incorporated byreference.

In a preferred embodiment, n and p are 0, and R₁, R₂, R₃, R₄, and R₅ arehydrogen. In another preferred embodiment, n and p are 0; R₁, R₂, R₃,R₄, and R₅ are hydrogen; and R⁶ is hexyl, butyl, ethyl, or phenylethyl.

In a further preferred embodiment, the compound has the structure:

In the above-described method of inhibiting the formation of a cataractin an eye, the eye may already contain one or more developing or fullydeveloped cataracts before it is contacted with the compound.Accordingly, the above-described method can be used to inhibit theformation of further cataracts in the eye, or to inhibit the formationof mature cataracts from the developing cataracts already present in theeye. Alternatively, the eye may be free of any developing or fullydeveloped cataracts before it is contacted with the compound.

As used herein, the term “contacting” is intended to encompass anymethod of directly applying the compound to the eye. In theabove-described method, any suitable means known to those of ordinaryskill in the art may be used to contact the eye with the compound.Examples of such methods include, but are not limited to, the compoundbeing injected into the eye, or being dropped or sprayed into the eye,or otherwise topically applied to the eye.

As used herein, the term “effective cataract-inhibiting amount” isintended to mean any amount which will inhibit the progression orformation of cataracts in an eye or inhibit the progression or formationof mature cataracts from any developing cataracts already present in theeye. The effective cataract-inhibiting amount of the compound willdepend on various factors known to those of ordinary skill in the art.Such factors include, but are not limited to, the size of the eye, andthe number and progression of any fully developed or developingcataracts already present in the eye. The effective cataract-inhibitingamount will also depend on whether the eye is to be contacted a singletime with the compound or whether the eye is to be contacted with thecompound periodically, over a stretch of time. The stretch of time maybe any number of days, weeks, months, or years. In one embodiment, theeffective cataract-inhibiting amount of the compound may be betweenabout 1.0 μg and 20.0 μg per eye. Preferably, the effectivecataract-inhibiting amount is between about 1.0 μg and 10.0 μg per eye.More preferably, the effective cataract-inhibiting amount of thecompound is between about 2.0 μg and about 5.0 μg per eye, this amountbeing especially preferred when the eye is contacted with the compoundperiodically, over a stretch of time.

This invention also provides a method of inhibiting the formation of acataract in an eye of a subject which comprises administering to thesubject a pharmaceutical composition which comprises a pharmaceuticallyacceptable carrier and an effective cataract-inhibiting amount of acompound having the structure:

wherein

X and Y are independently carbonyl, C═S, S═O, or O═S═O; n and p areindependently 0 or 1;

R¹ and R² are independently hydrogen, linear or branched C₁-C₁₈ alkyl,alkenyl, or alkynyl, either unsubstituted or substituted with halogen,including F, Cl, Br, and I, C₁-C₆ alkoxy, (C═O)R⁷, or (C═O)OR⁸; whereinR⁷ and R¹ are independently C₁-C₆ linear or branched alkyl;

R³, R⁴, and R⁵ are independently hydrogen, halogen, including F, Cl, Br,and I, trihalomethyl, C₁-C₁₈ linear or branched alkyl, alkenyl, alkynyl,haloalkyl, alkoxy, alkoxyalkyl, alkylthio, or (C═O)R⁹, (C═O)OR¹⁰,O(C═O)R¹¹, (C═S)R¹², (C═S)OR¹³, O(C═S)R¹⁴, (S═O)R¹⁵, (S═O)OR¹⁶, or(O═S═O)OR¹⁷; wherein R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ areindependently C₁-C₁₈ linear or branched alkyl, alkenyl, or alkynyl; and

R⁶ is aryl or C₁-C₁₈ branched or linear alkyl, alkenyl, or alkynyl,either unsubstituted or substituted with halogen, including, F, Cl, Br,and I, C₁-C₆ alkoxy, (C═O)R¹⁸, (C═O)OR¹⁹, or aryl; wherein R¹⁸ and R¹⁹are independently C₁-C₆ branched or linear alkyl;

or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, n and p are 0, and R₁, R₂, R₃, R₄, and R₅ arehydrogen. In another preferred embodiment, n and p are 0; R₁, R₂, R₃,R₄, and R₅ are hydrogen; and R₆ is hexyl, butyl, ethyl, or phenylethyl.

In a further preferred embodiment, the compound has the structure:

In the above-described method of inhibiting the formation of a cataractin an eye of a subject, the eye may already contain one or moredeveloping or fully developed cataracts before the pharmaceuticalcomposition is administered to the subject. Accordingly, theabove-described method can be used to inhibit the formation of furthercataracts in the eye of the subject, or to inhibit the formation ofmature cataracts from the developing cataracts already present in theeye of the subject. Alternatively, the eye of the subject may be free ofany developing or fully developed cataracts before the pharmaceuticalcomposition is administered to the subject.

In the above-described method, any suitable means known to those ofordinary skill in the art may be used to administer the pharmaceuticalcomposition to the subject. In one embodiment, administering thepharmaceutical composition to the subject comprises applying thepharmaceutical composition to the eye of the subject.

In another embodiment, the pharmaceutical composition is orallyadministered to the subject. If oral administration is employed, thepharmaceutical composition may be in the form of a capsule, tablet, orsolution.

In another embodiment, the pharmaceutical composition is injected intothe subject. Injection may be intramuscular, intraperitoneal,intravenous, or subcutaneous. The pharmaceutical composition may beinjected into any part of the subject's body, including into one or bothof the subject's eyes.

In a further embodiment, the pharmaceutical composition is topicallyapplied to the subject. If the pharmaceutical composition is topicallyapplied, the pharmaceutical composition may be in the form of a lotionor cream. The pharmaceutical composition may be topically applied to anypart of the subject's body, since topical administration will result insystemic effects. If the pharmaceutical composition is topically appliedto one or both of the eyes of the subject, the pharmaceuticalcomposition may be in the form of eye drops.

Finally, the administration may comprise surgically removing the lens ofthe eye from the subject, applying the pharmaceutical composition to thelens, and then surgically replacing the lens.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutically accepted carriers knownto those of ordinary skill in the art. Examples of such standardcarriers include, but are not limited to, phosphate buffered salinesolution, water, emulsions such as oil/water emulsions or a triglycerideemulsion, various types of wetting agents, tablets, coated tablets andcapsules. A suitable pharmaceutically accepted carrier may be selectedtaking into account the chosen mode of administration.

As used herein, the term “effective cataract-inhibiting amount” isintended to mean any amount which will inhibit the progression orformation of cataracts in an eye or inhibit the progression or formationof mature cataracts from any developing cataracts already present in theeye. The effective cataract-inhibiting amount of the compound willdepend on various factors known to those of ordinary skill in the art.Such factors include, but are not limited to, the size of the eye, thenumber and progression of any fully developed or developing cataractsalready present in the eye, and the mode of administration. Theeffective cataract-inhibiting amount will also depend on whether thepharmaceutical composition is to be administered a single time, orwhether the pharmaceutical composition is to be administeredperiodically, over a stretch of time. The stretch of time may be anynumber of days, weeks, months, or years. In one embodiment, theeffective cataract-inhibiting amount of the compound may be betweenabout 1.0 μg and 20.0 μg. Preferably, the effective cataract-inhibitingamount is between about 1.0 μg and 10.0 μg.

More preferably, the effective cataract-inhibiting amount of thecompound is between about 2.0 μg and about 5.0 μg, this amount beingespecially preferred when the pharmaceutical composition is applied toone or both of the eyes of the subject, periodically, over a stretch oftime.

In one embodiment of the above-described method of inhibiting theformation of a cataract in an eye of a subject, the subject is a mammal.When the subject is a mammal, the subject may be a human being.

This invention also provides a method of inhibiting the progression ofcataract formation in an eye which comprises contacting the eye with aneffective cataract-inhibiting amount of a compound having the structure:

wherein

X and Y are independently carbonyl, C═S, S═O, or O═S═O; n and p areindependently 0 or 1;

R¹ and R² are independently hydrogen, linear or branched C₁-C₁₈ alkyl,alkenyl, or alkynyl, either unsubstituted or substituted with halogen,including F, Cl, Br, and I, C₁-C₆ alkoxy, (C═O)R⁷, or (C═O)OR⁸; whereinR⁷ and R⁸ are independently C₁-C₆ linear or branched alkyl;

R³, R⁴, and R⁵ are independently hydrogen, halogen, including F, Cl, Br,and I, trihalomethyl, C₁-C₁₈ linear or branched alkyl, alkenyl, alkynyl,haloalkyl, alkoxy, alkoxyalkyl, alkylthio, or (C═O)R⁹, (C═O)OR¹⁰,O(C═O)R¹¹, (C═S)R¹², (C═S)OR¹³, O(C═S)R¹⁴, (S═O)R¹⁵, (S═O)OR¹⁶, or(O═S═O)OR¹⁷; wherein R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ areindependently C₁-C₁₈ linear or branched alkyl, alkenyl, or alkynyl; and

R⁶ is aryl or C₁-C₁₈ branched or linear alkyl, alkenyl, or alkynyl,either unsubstituted or substituted with halogen, including, F, Cl, Br,and I, C₁-C₆ alkoxy, (C═O)R¹⁸, (C═O)OR¹⁹, or aryl; wherein R¹⁸ and R¹⁹are independently C₁-C₆ branched or linear alkyl;

or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, n and p are 0, and R₁, R₂, R₃, R₄, and R₅ arehydrogen. In another preferred embodiment, n and p are 0; R₁, R₂, R₃,R₄, and R₅ are hydrogen; and R₆ is hexyl, butyl, ethyl, or phenylethyl.

In a further preferred embodiment, the compound has the structure:

In the above-described method of inhibiting the progression of cataractformation in an eye, the eye may already contain one or more developingor fully developed cataracts before it is contacted with the compound.Accordingly, the above-described method can be used to inhibit theprogression of cataract formation from any developing cataracts alreadypresent in the eye. Alternatively, the eye may be free of any developingor fully developed cataracts.

As used herein, the term “contacting” is intended to encompass anymethod of directly applying the compound to the eye. In theabove-described method, any suitable means known to those of ordinaryskill in the art may be used to contact the eye with the compound.Examples of such methods include, but are not limited to, the compoundbeing injected into the eye, or being dropped or sprayed into the eye,or otherwise topically applied to the eye.

As used herein, the term “effective cataract-inhibiting amount” isintended to mean any amount which will inhibit the progression orformation of cataracts in an eye or inhibit the progression or formationof mature cataracts from any developing cataracts already present in theeye. The effective cataract-inhibiting amount of the compound willdepend on various factors known to those of ordinary skill in the art.Such factors include, but are not limited to, the size of the eye, andthe number and progression of any fully developed or developingcataracts already present in the eye. The effective cataract-inhibitingamount will also depend on whether the eye is to be contacted a singletime with the compound or whether the eye is to be contacted with thecompound periodically, over a stretch of time. The stretch of time maybe any number of days, weeks, months, or years. In one embodiment, theeffective cataract-inhibiting amount of the compound may be betweenabout 1.0 μg and 20.0 μg per eye. Preferably, the effectivecataract-inhibiting amount is between about 1.0 μg and 10.0 μg per eye.More preferably, the effective cataract-inhibiting amount of thecompound is between about 2.0 μg and about 5.0 μg per eye, this amountbeing especially preferred when the eye is contacted with the compoundperiodically, over a stretch of time.

Still further, this invention provides a method of inhibiting theprogression of cataract formation in an eye of a subject which comprisesadministering to the subject a pharmaceutical composition whichcomprises a pharmaceutically acceptable carrier and an effectivecataract-inhibiting amount of a compound having the structure:

wherein

X and Y are independently carbonyl, C═S, S═O, or O═S═O; n and p areindependently 0 or 1;

R¹ and R² are independently hydrogen, linear or branched C₁-C₁₈ alkyl,alkenyl, or alkynyl, either unsubstituted or substituted with halogen,including F, Cl, Br, and I, C₁-C₆ alkoxy, (C═O)R⁷, or (C═O)OR⁸; whereinR⁷ and R⁸ are independently C₁-C₆ linear or branched alkyl;

R³, R⁴, and R⁵ are independently hydrogen, halogen, including F, Cl, Br,and I, trihalomethyl, C₁-C₁₈ linear or branched alkyl, alkenyl, alkynyl,haloalkyl, alkoxy, alkoxyalkyl, alkylthio, or (C═O)R⁹, (C═O)OR¹⁰,(C═O)R¹¹, (C═S)R¹², (C═S)OR¹³, O(C═S)R¹⁴, (S═O)R¹⁵, (S═O)OR¹⁶, or(O═S═O)OR¹⁷; wherein R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ areindependently C₁-C₁₈ linear or branched alkyl, alkenyl, or alkynyl; and

R⁶ is aryl or C₁-C₁₈ branched or linear alkyl, alkenyl, or alkynyl,either unsubstituted or substituted with halogen, including, F, Cl, Br,and I, C₁-C₆ alkoxy, (C═O)R¹⁸, (C═O)OR¹⁹, or aryl; wherein R¹⁸ and R¹⁹are independently C₁-C₆ branched or linear alkyl;

or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, n and p are 0, and R₁, R₂, R₃, R₄, and R₅ arehydrogen. In another preferred embodiment, n and p are 0; R₁, R₂, R₃,R₄, and R₅ are hydrogen; and R₆ is hexyl, butyl, ethyl, or phenylethyl.

In a further preferred embodiment, the compound has the structure:

In the above-described method of inhibiting the progression of cataractformation in an eye of a subject, the eye may already contain one ormore developing or fully developed cataracts before the pharmaceuticalcomposition is administered to the subject. Accordingly, theabove-described method can be used to inhibit the progression ofcataract formation from any developing cataracts already present in theeye of the subject. Alternatively, the eye of the subject may be free ofany developing or fully developed cataracts before the pharmaceuticalcomposition is administered to the subject.

In the above-described method, any suitable means known to those ofordinary skill in the art may be used to administer the pharmaceuticalcomposition to the subject. In one embodiment, administering thepharmaceutical composition to the subject comprises applying thepharmaceutical composition to the eye of the subject.

In another embodiment, the pharmaceutical composition is orallyadministered to the subject. If oral administration is employed, thepharmaceutical composition may be in the form of a capsule, tablet, orsolution.

In another embodiment, the pharmaceutical composition is injected intothe subject. Injection may be intramuscular, intraperitoneal,intravenous, or subcutaneous. The pharmaceutical composition may beinjected into any part of the subject's body, including into one or bothof the subject's eyes.

In a further embodiment, the pharmaceutical composition is topicallyapplied to the subject. If the pharmaceutical composition is topicallyapplied, the pharmaceutical composition may be in the form of a lotionor cream. The pharmaceutical composition may be topically applied to anypart of the subject's body, since topical administration will result insystemic effects. If the pharmaceutical composition is topically appliedto one or both of the eyes of the subject, the pharmaceuticalcomposition may be in the form of eye drops.

Finally, the administration may comprise surgically removing the lens ofthe eye from the subject, applying the pharmaceutical composition to thelens, and then surgically replacing the lens.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutically accepted carriers knownto those of ordinary skill in the art. Examples of such standardcarriers include, but are not limited to, phosphate buffered salinesolution, water, emulsions such as oil/water emulsions or a triglycerideemulsion, various types of wetting agents, tablets, coated tablets andcapsules. A suitable pharmaceutically accepted carrier may be selectedtaking into account the chosen mode of administration.

As used herein, the term “effective cataract-inhibiting amount” isintended to mean any amount which will inhibit the progression orformation of cataracts in an eye or inhibit the progression or formationof mature cataracts from any developing cataracts already present in theeye. The effective cataract-inhibiting amount of the compound willdepend on various factors known to those of ordinary skill in the art.Such factors include, but are not limited to, the size of the eye, thenumber and progression of any fully developed or developing cataractsalready present in the eye, and the mode of administration. Theeffective cataract-inhibiting amount will also depend on whether thepharmaceutical composition is to be administered a single time, orwhether the pharmaceutical composition is to be administeredperiodically, over a stretch of time. The stretch of time may be anynumber of days, weeks, months, or years. In one embodiment, theeffective cataract-inhibiting amount of the compound may be betweenabout 1.0 μg and 20.0 μg per eye. Preferably, the effectivecataract-inhibiting amount is between about 1.0 μg and 10.0 μg. Morepreferably, the effective cataract-inhibiting amount of the compound isbetween about 2.0 μg and about 5.0 μg, this amount being especiallypreferred when the pharmaceutical composition is applied to one or bothof the eyes of the subject, periodically, over a stretch of time.

In one embodiment of the above-described method of inhibiting theprogression of cataract formation in an eye of a subject, the subject isa mammal. When the subject is a mammal, the subject may be a humanbeing.

This invention further provides a method of preventing in a subject adisease resulting from oxidative stress which comprises administering tothe subject a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and an effective oxidative stress-inhibiting amountof a compound having the structure:

wherein

X and Y are independently carbonyl, C═S, S═O, or O═S═O; n and p areindependently 0 or 1;

R¹ and R² are independently hydrogen, linear or branched C₁-C₁₈ alkyl,alkenyl, or alkynyl, either unsubstituted or substituted with halogen,including F, Cl, Br, and I, C₁-C₆ alkoxy, (C═O)R⁷, or (C═O)OR⁸; whereinR⁷ and R⁸ are independently C₁-C₆ linear or branched alkyl;

R³, R⁴, and R⁵ are independently hydrogen, halogen, including F, Cl, Br,and I, trihalomethyl, C₁-C₁₈ linear or branched alkyl, alkenyl, alkynyl,haloalkyl, alkoxy, alkoxyalkyl, alkylthio, or (C═O)R⁹, (C═O)OR¹⁰,O(C═O)R¹¹, (C═S)R¹², (C═S)OR¹³, O(C═S)R¹⁴, (S═O)R¹⁵, (S═O)OR¹⁶, or(O═S═O)OR¹⁷; wherein R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ areindependently C₁-C₁₈ linear or branched alkyl, alkenyl, or alkynyl; and

R⁶ is aryl or C₁-C₁₈ branched or linear alkyl, alkenyl, or alkynyl,either unsubstituted or substituted with halogen, including, F, Cl, Br,and I, C₁-C₆ alkoxy, (C═O)R¹⁸, (C═O)OR¹⁹, or aryl; wherein R¹⁸ and R¹⁹are independently C₁-C₆ branched or linear alkyl;

or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, n and p are 0, and R₁, R₂, R₃, R₄, and R₅ arehydrogen. In another preferred embodiment, n and p are 0; R₁, R₂, R₃,R₄, and R₅ are hydrogen; and R₆ is hexyl, butyl, ethyl, or phenylethyl.

In a further preferred embodiment, the compound has the structure:

As used herein, a disease resulting from oxidative stress is any diseaseresulting from the effects of reactive oxygen species generated incells. Reactive oxygen species include superoxide, H₂O₂, and hydroxylradicals. Reactive oxygen species may be generated in cells duringaerobic cellular metabolism. Also, certain external agents are known toresult in the generation of reactive oxygen species in cells, andthereby in an increase in oxidative stress. External agents which mayresult in the generation of reactive oxidative species in cells includecertain types of radiation, such as UV radiation or X-rays,chemotherapeutic agents, pesticides, and cigarette smoke. Metabolizingxenobiotics or fatty foods may also result in generation of reactiveoxygen species in cells.

Diseases which may result from oxidative stress are known to those ofordinary skill in the art, and include, but are not limited to,rheumatoid arthritis, lupus, and sickle cell anemia. Oxidative stresscauses oxidation of DNA bases and damages DNA in cells, and consequentlymay also lead to the formation of tumors. Thus, diseases which resultfrom oxidative stress include diseases comprising the formation oftumors resulting from oxidative stress. Diseases which comprise theformation of tumors include cancer.

In the above-described method, any suitable means known to those ofordinary skill in the art may be used to administer the pharmaceuticalcomposition to the subject. In one embodiment, the pharmaceuticalcomposition is topically applied to a part of the subject's body whereoxidative stress is expected. However, topical application of thepharmaceutical composition to one part of the subject's body can beexpected to have beneficial systemic effects on other parts. When thecomposition is topically applied, it may be in the form of a cream or alotion.

In another embodiment, the pharmaceutical composition is orallyadministered to the subject. If oral administration is employed, thepharmaceutical composition may be in the form of a capsule, tablet, orsolution.

In another embodiment, the pharmaceutical composition may be injectedinto the subject. Injection may be intramuscular, intraperitoneal,intravenous, or subcutaneous. The pharmaceutical composition may beinjected into any part of the subject's body, including into a partwhere oxidative stress is expected. As used herein, the term“pharmaceutically acceptable carrier” encompasses any of the standardpharmaceutically accepted carriers known to those of ordinary skill inthe art. Examples of such standard carriers include, but are not limitedto, phosphate buffered saline solution, water, emulsions such asoil/water emulsions or a triglyceride emulsion, various types of wettingagents, tablets, coated tablets and capsules. A suitablepharmaceutically accepted carrier may be selected taking into accountthe chosen mode of administration.

As used herein, the term “effective oxidative stress-inhibiting amount”is meant to indicate that amount which will inhibit the generation ofreactive oxygen species in cells. The effective oxidativestress-inhibiting amount of the compound will depend on various factorsknown to those of ordinary skill in the art. Such factors include, butare not limited to, the size of the subject and the mode ofadministration. The effective oxidative stress-inhibiting amount of thecompound will also depend on whether the pharmaceutical composition isto be administered a single time to the subject, or whether it is to beadministered periodically over a stretch of time. A stretch of time maybe any number of days, weeks, months, or years. In one embodiment, theoxidative stress-inhibiting amount of the compound may be between about0.01 μg and about 20.0 μg per dose. Preferably the oxidativestress-inhibiting amount of the compound is between about 0.01 μg andabout 10.0 μg per dose. Even more preferably, the oxidativestress-inhibiting amount of the compound is between about 0.02 μg and2.0 μg per dose, this amount being especially preferred when thepharmaceutical composition is administered periodically, over a stretchof time.

In one embodiment of the above-described method of preventing in asubject a disease resulting from oxidative stress, the subject is amammal. When the subject is a mammal, the subject may be a human being.

Further provided is a method of inhibiting in a subject the progressionof a disease resulting from oxidative stress which comprisesadministering to the subject a pharmaceutical composition comprising apharmaceutically acceptable carrier and an effective oxidativestress-inhibiting amount of a compound having the structure:

wherein

X and Y are independently carbonyl, C═S, S═O, or O═S═O; n and p areindependently 0 or 1;

R¹ and R² are independently hydrogen, linear or branched C₁-C₁₈ alkyl,alkenyl, or alkynyl, either unsubstituted or substituted with halogen,including F, Cl, Br, and I, C₁-C₆ alkoxy, (C═O)R⁷, or (C═O)OR⁸; whereinR⁷ and R⁸ are independently C₁-C₆ linear or branched alkyl;

R³, R⁴, and R⁵ are independently hydrogen, halogen, including F, Cl, Br,and I, trihalomethyl, C₁-C₁₈ linear or branched alkyl, alkenyl, alkynyl,haloalkyl, alkoxy, alkoxyalkyl, alkylthio, or (C═O)R⁹, (C═O)OR¹⁰,O(C═O)R¹¹, (C═S)R¹², (C═S)OR¹³, O(C═S)R¹⁴, (S═O)R¹⁵, (S═O)OR¹⁶, or(O═S═O)OR¹⁷; wherein R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ areindependently C₁-C₁₈ linear or branched alkyl, alkenyl, or alkynyl; and

R⁶ is aryl or C₁-C₁₈ branched or linear alkyl, alkenyl, or alkynyl,either unsubstituted or substituted with halogen, including, F, Cl, Br,and I, C₁-C₆ alkoxy, (C═O)R¹⁸, (C═O)OR¹⁹, or aryl; wherein R¹⁸ and R¹⁹are independently C₁-C₆ branched or linear alkyl;

or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, n and p are 0, and R₁, R₂, R₃, R₄, and R₅ arehydrogen. In another preferred embodiment, n and p are 0; R₁, R₂, R₃,R₄, and R₅ are hydrogen; and R₆ is hexyl, butyl, ethyl, or phenylethyl.

In further preferred embodiment, the compound has the structure:

As described above, a disease resulting from oxidative stress is anydisease resulting from the effects of reactive oxygen species generatedin cells. By inhibiting generation of reactive oxygen species in thecells of a subject already afflicted with a disease resulting fromoxidative stress, the progression of the disease may be inhibited. Asdescribed above, diseases which may result from oxidative stress includerheumatoid arthritis, lupus, and sickle cell anemia. The progression ofthe aforementioned diseases may therefore be inhibited by employing thesubject method. Likewise, since oxidative stress results in damage tothe DNA in cells, which consequently may result in the formation oftumors, the progression of diseases comprising the formation of tumorsresulting from oxidative stress may also be inhibited by employing thesubject method. Since the formation of tumors resulting from oxidativestress involves damage to DNA in cells, tumor formation resulting fromoxidative stress includes tumors evolving from normal tissue as well asthe enlargement of already developing tumors by damage to the DNA incells surrounding the already developing tumor. As described above,diseases which comprise the formation of tumors include cancer.

In the above-described method, any suitable means known to those ofordinary skill in the art may be used to administer the pharmaceuticalcomposition to the subject. In one embodiment, the pharmaceuticalcomposition is topically applied to a part of the subject's body alreadymanifesting the disease resulting from oxidative stress, e.g. to atumor. However, topical application of the pharmaceutical composition toone part of the subject's body can be expected to have beneficialsystemic effects on other parts, and thus the pharmaceutical compositionneed not be applied to the part of the subject's body manifesting thedisease resulting from oxidative stress, should such a part exist. Whenthe composition is topically applied, it may be in the form of a creamor a lotion.

In another embodiment, the pharmaceutical composition is orallyadministered to the subject. If oral administration is employed, thepharmaceutical composition may be in the form of a capsule, tablet, orsolution.

In another embodiment, the pharmaceutical composition may be injectedinto the subject. Injection may be intramuscular, intraperitoneal,intravenous, or subcutaneous. The pharmaceutical composition may beinjected into any part of the subject's body, including into a partmanifesting the disease resulting from oxidative stress, e.g. a tumor.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutically accepted carriers knownto those of ordinary skill in the art. Examples of such standardcarriers include, but are not limited to, phosphate buffered salinesolution, water, emulsions such as oil/water emulsions or a triglycerideemulsion, various types of wetting agents, tablets, coated tablets andcapsules. A suitable pharmaceutically accepted carrier may be selectedtaking into account the chosen mode of administration.

As used herein, the term “effective oxidative stress-inhibiting amount”is meant to indicate that amount which will inhibit the generation ofreactive oxygen species in cells. The effective oxidativestress-inhibiting amount of the compound will depend on various factorsknown to those of ordinary skill in the art. Such factors include, butare not limited to, the size of the subject and the mode ofadministration. The effective oxidative stress-inhibiting amount of thecompound will also depend on whether the pharmaceutical composition isto be administered a single time to the subject, or whether it is to beadministered periodically over a stretch of time. A stretch of time maybe any number of days, weeks, months, or years. In one embodiment, theoxidative stress-inhibiting amount of the compound may be between about0.01 μg and about 20.0 μg per dose. Preferably the oxidativestress-inhibiting amount of the compound is between about 0.01 μg andabout 10.0 μg per dose. Even more preferably, the oxidativestress-inhibiting amount of the compound is between about 0.02 μg and2.0 μg per dose, this amount being especially preferred when thepharmaceutical composition is administered periodically, over a stretchof time.

In one embodiment of the above-described method of preventing in asubject a disease resulting from oxidative stress, the subject is amammal. When the subject is a mammal, the subject may be a human being.

This invention further provides a method of treating a subject having anHIV infection which comprises administering to the subject apharmaceutical composition comprising a pharmaceutically acceptablecarrier, a substance which inhibits HIV replication, and an effectiveHIV integrase-inhibiting amount of a compound having the structure:

wherein

X and Y are independently carbonyl, C═S, S═O, or O═S═O; n and p areindependently 0 or 1;

R¹ and R² are independently hydrogen, linear or branched C₁-C₁₈ alkyl,alkenyl, or alkynyl, either unsubstituted or substituted with halogen,including F, Cl, Br, and I, C₁-C₆ alkoxy, (C═O)R⁷, or (C═O)OR⁸; whereinR⁷ and R⁸ are independently C₁-C₆ linear or branched alkyl;

R³, R⁴, and R₅ are independently hydrogen, halogen, including F, Cl, Br,and I, trihalomethyl, C₁-C₁₈ linear or branched alkyl, alkenyl, alkynyl,haloalkyl, alkoxy, alkoxyalkyl, alkylthio, or (C═O)R⁹, (C═O)OR¹⁰,O(C═O)R¹¹, (C═S)R¹², (C═S)OR¹³, O(C═S)R¹⁴, (S═O)R¹⁵, (S═O)OR¹⁶, or(O═S═O)OR¹⁷; wherein R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ areindependently C₁-C₁₈ linear or branched alkyl, alkenyl, or alkynyl; and

R⁶ is aryl or C₁-C₁₈ branched or linear alkyl, alkenyl, or alkynyl,either unsubstituted or substituted with halogen, including, F, Cl, Br,and I, C₁-C₆ alkoxy, (C═O)R¹⁸, (C═O)OR¹⁹, or aryl; wherein R¹⁸ and R¹⁹are independently C₁-C₆ branched or linear alkyl;

or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, n and p are 0, and R₁, R₂, R₃, R₄, and R₅ arehydrogen. In another preferred embodiment, n and p are 0; R₁, R₂, R₃,R₄, and R₅ are hydrogen; and R₆ is hexyl, butyl, ethyl, or phenylethyl.

In a further preferred embodiment, the compound has the structure:

As the compound in the above-described method inhibits HIV integrase,the substance which inhibits HIV replication is preferably a substancewhich inhibits an enzyme in the HIV replication cycle other than HIVintegrase. Preferably, the substance which inhibits HIV replicationinhibits HIV reverse transcriptase. If the substance which inhibits HIVreplication is a substance which inhibits HIV reverse transcriptase, itis preferably 3′-azido-3′-deoxythymidine.

In the above-described method, any suitable means known to those ofordinary skill in the art may be used to administer the pharmaceuticalcomposition to the subject. In one embodiment, the pharmaceuticalcomposition is topically applied to the subject. If the pharmaceuticalcomposition is topically applied, the pharmaceutical composition may bein the form of a lotion or a cream. In another embodiment, thepharmaceutical composition is orally administered to the subject. If thepharmaceutical composition is orally administered, it may be in the formof a tablet, capsule, or solution. In another embodiment, thepharmaceutical composition is injected into the subject. The injectionmay be intravenous, intraperitoneal, intramuscular, or subcutaneous.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutically accepted carriers knownto those of ordinary skill in the art. Examples of such standardcarriers include, but are not limited to, phosphate buffered salinesolution, water, emulsions such as oil/water emulsions or a triglycerideemulsion, various types of wetting agents, tablets, coated tablets andcapsules. A suitable pharmaceutically accepted carrier may be selectedtaking into account the chosen mode of administration.

As used herein, the term “effective HIV integrase-inhibiting amount” isintended to mean that amount which is effective to inhibit HIVintegrase. The effective HIV integrase-inhibiting amount will depend onvarious factors known to those of ordinary skill in the art. Suchfactors include, but are not limited to, the size of the subject and thedegree to which the HIV infection has already progressed in the subject.The effective HIV integrase-inhibiting amount of the compound will alsodepend on whether the pharmaceutical composition is to be administeredto the subject a single time, or whether the pharmaceutical compositionis to be administered periodically over a stretch of time.

In one embodiment of the above-described method of treating a subjecthaving an HIV infection, the subject is a mammal. When the subject is amammal, the subject may be a human being.

Finally, this invention provides a pharmaceutical composition fortreating an HIV infection in a subject which comprises apharmaceutically acceptable carrier, a substance which inhibits HIVreplication, and an effective HIV integrase-inhibiting amount of acompound having the structure:

wherein

X and Y are independently carbonyl, C═S, S═O, or O═S═O; n and p areindependently 0 or 1;

R¹ and R² are independently hydrogen, linear or branched C₁-C₁₈ alkyl,alkenyl, or alkynyl, either unsubstituted or substituted with halogen,including F, Cl, Br, and I, C₁-C₆ alkoxy, (C═O)R⁷, or (C═O)OR⁸; whereinR⁷ and R⁸ are independently C₁-C₆ linear or branched alkyl;

R³, R⁴, and R⁵ are independently hydrogen, halogen, including F, Cl, Br,and I, trihalomethyl, C₁-C₁₈ linear or branched alkyl, alkenyl, alkynyl,haloalkyl, alkoxy, alkoxyalkyl, alkylthio, or (C═O)R⁹, (C═O)OR¹⁰,O(C═O)R¹¹, (C═S)R¹², (C═S)OR¹³, O(C═S)R¹⁴, (S═O)R¹⁵, (S═O)OR¹⁶, or(O═S═O)OR¹⁷; wherein R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ areindependently C₁-C₁₈ linear or branched alkyl, alkenyl, or alkynyl; and

R⁶ is aryl or C₁-C₁₈ branched or linear alkyl, alkenyl, or alkynyl,either unsubstituted or substituted with halogen, including, F, Cl, Br,and I, C₁-C₆ alkoxy, (C═O)R¹⁸, (C═O)OR¹⁹, or aryl; wherein R¹⁸ and R¹⁹are independently C₁-C₆ branched or linear alkyl;

or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, n and p are 0, and R₁, R₂, R₃, R⁴, and R₅ arehydrogen. In another embodiment, n and p are 0; R₁, R₂, R₃, R₄, and R₅are hydrogen; and R₆ is hexyl, butyl, ethyl, or phenylethyl.

In a further preferred embodiment, the compound has the structure:

Preferably, the substance which inhibits HIV replication is a substancewhich inhibits HIV reverse transcriptase. If the substance whichinhibits HIV replication inhibits HIV reverse transcriptase, it ispreferably 3′-azido-3′-deoxythymidine.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutically accepted carriers knownto those of ordinary skill in the art. Examples of such standardcarriers include, but are not limited to, phosphate buffered salinesolution, water, emulsions such as oil/water emulsions or a triglycerideemulsion, various types of wetting agents, tablets, coated tablets andcapsules. A suitable pharmaceutically accepted carrier may be selectedtaking into account the chosen mode of administration.

As used herein, the term “effective HIV integrase-inhibiting amount” isintended to mean that amount which is effective to inhibit HIVintegrase. The effective HIV integrase-inhibiting amount will depend onvarious factors known to those of ordinary skill in the art. Suchfactors include, but are not limited to, the size of the subject, thedegree to which the HIV infection has already progressed in the subject,and the pharmaceutically accepted carrier in the composition. Theeffective HIV integrase-inhibiting amount of the compound will alsodepend on whether the pharmaceutical composition is to be administeredto the subject a single time, or whether the pharmaceutical compositionis to be administered periodically over a stretch of time.

This invention will be better understood from the Examples in the“Experimental Details” Section which follows. However, one skilled inthe art will readily appreciate that the specific methods and resultsdiscussed are merely illustrative of, and are not intended to, norshould they be intended to, limit the invention as described more fullyin the claims which follow thereafter.

EXPERIMENTAL DETAILS EXAMPLE 1

Chemicals and Reagents for In-Vivo Experiments on Mice and In VitroExperiments

CAPE was obtained by esterification of caffeic acid with phenethylalcohol in the presence of p-toluenesulfonic acid as the catalyst (1).A.S.A.P. DNA purification columns and the enzymes used for purificationand enzymatic hydrolysis of DNA (proteinase K, RNase, DNase I, nucleaseP₁, and alkaline phosphatase) were purchased from Boehringer-MannheimBiochemicals (Indianapolis, Ind.). Other reagents and enzymes (such asglucose, glucose oxidase, horseradish peroxidase (HRPO), and2′,7′-dichlorofluorescin diacetate) were purchased from Sigma ChemicalCo. (St. Louis, Mo.), and HPLC-grade acetonitrile and acetone were fromFisher Scientific (Springfield, N.J.).

EXAMPLE 2

Radioactive Materials For In Vivo Experiments on Mice and In VitroExperiments

[³H]acetic anhydride (specific activity, 50 mCi/mmol) was obtained fromDu Pont New England Nuclear (Wilmington, Del.). Nonradioactive caffeicacid, HMdUrd, and TPA were purchased from Sigma. TPA was also purchasedfrom CRC, Inc. (Chanhassen, Minn.). 8-OHdGua was synthesized accordingto published procedures (49, 50), while the marker acetates wereprepared by acetylation of HMdUrd and 8-OHdGua with acetic anhydride inthe presence of a catalyst in dry acetonitrile (49, 51). The productswere purified by high performance liquid chromatography (HPLC) and theiridentity confirmed by mass spectroscopy (50).

EXAMPLE 3

In Vivo Experiments on Mice

A) Treatment of Animals and Assays

Female SENCAR (6-8 weeks old) and CD-1 mice (about 7 weeks old) werepurchased from Biological Testing, National Cancer Institute (Frederick,Md.) and from Charles River Laboratory (Stone Ridge, N.Y.),respectively. The mice were acclimated for at least 1 week before use.They were subjected to a 12-h light/12-h dark cycle, housed at 20±2° C.in a room with 10 cycles of air exchanges/h, given food and water adlibitum, and observed for any indication of ill health. Before topicalapplication of any test agents, their backs were shaved and only thosewithout hair growth after 48 h were used.

B) Edema Determination

Both ears of SENCAR mice (7-9 mice/group) were treated with 0.4 nmol TPAin 20 μl acetone alone or together with added 0.1 nmol, 0.3 nmol, or 1μmol CAPE. Control mice were treated with acetone. The mice weresacrificed by cervical dislocation 5 h after treatment, and edema wasmeasured on one mouse ear, while the second ear was immediately frozenand later analyzed for PMN infiltration by quantifying myeloperoxidase(MPO) (see below). Ear edema (increased weight of ear punches) wasdetermined as previously described (24). Edema was expressed as mg/6 mm(diameter) ear punch±SE.

C) MPO, H₂O₂, and Oxidized DNA Bases

SENCAR mice were pretreated with CAPE for 30 min; then 6.5 nmol (4 μg)TPA/mouse topically applied to the dorsal skin. After 20 h, the CAPE andTPA treatment (1×) was repeated (2× treatment). One h after the secondtreatment, the mice were sacrificed by cervical dislocation, and 12.5-mmpunches were obtained to be used separately for MPO and H₂O₂determinations. The remainder of the treated mouse skin or the combinedskins from two mice were used for separation of epidermal cells. Thiswas done by heating the skin at 55° C. for 30 s followed by rapidimmersion into an ice-cold water bath (45). After the epidermis wasscraped off, the presence of oxidized DNA base derivatives was measuredin the epidermal DNA. All of these end points were analyzed by publishedprocedures and are briefly described below.

The assay for MPO was carried out according to the procedure of Metcalfet al. (53) with some modifications (20), and the results are expressedas units/cm², where a unit is defined as that degrading 1 μmol H₂O₂/minat 25° C. The presence of MPO in the mouse ears was measured in thehomogenates. They were prepared by mincing the ears and homogenizingthem in an ice-cold water bath with a polytron homogenizer, either twicefor 20 s each or three times for 10 s each. Each homogenate was analyzedin duplicate and the results are expressed as mean MPO (units/mgprotein)±SE for each of the homogenates. In this case, SE showsreproducibility of each MPO analysis.

Punches used for H₂O₂ determination were immediately minced withscissors in cold 50 mM phosphate buffer, pH 7.0, containing 10 mM azidethat is needed for inhibition of catalase. They were then homogenized,and centrifuged at 4° C. and the supernatants were stored at −80° C. forup to 1 week. H₂O₂ was measured in freshly thawed supernatants usinghorseradish peroxidase-mediated oxidation of phenol red (21), and theresults are expressed as nmol H₂O₂/cm²/10-min incubation. Althoughorganic hydroperoxides also can be generated under these conditions(45), we find that H₂O₂ constitutes a great majority of the oxidantsgenerated because 60-85% of the oxidants produced are catalaseinhibitable (21).

The epidermis was homogenized and lysed, and the proteins and RNA wereremoved by proteinase K and RNase digestions, respectively (20). The DNAwas separated and purified on A.S.A.P. columns (Boehringer-MannheimBiochemicals) and precipitated with isopropyl alcohol.

The pellet was then washed with 70% ethyl alcohol, dried, and dissolvedin 10 mM Tris.HCl-100 mM NaCl buffer, pH 7.0, according to themanufacturer's procedure. The DNA was sheared and enzymatically digestedto nucleosides, and the hydrolysates were separated by HPLC (Beckman,Model 344) on an ODS column (Altex, 1×25 cm; 5 μm particle size) (50).The fractions that eluted after 30 ml were combined (excluding thosecontaining normal nucleosides) and dried. The oxidized nucleosides weredetected by ³H-postlabeling with [³H]acetic anhydride, followed by HPLCanalysis of ³H-containing acetates in the presence of marker acetates(50). The results are presented as the number of oxidizednucleosides/10⁴ normal bases, unless otherwise stated.

EXAMPLE 4

In Vitro Experiments

A) Human PMNs

Blood was obtained from healthy volunteers and collected intoEDTA-containing tubes. Red cells were removed by dextran sedimentationand lysis with hypotonic ammonium chloride, as described previously (5).Cells were washed in glucose-containing basic salt solution (137 mMNaCl, 5 mM KCl, 8.5 mM Na₂HPO₄-NaH₂PO₄, 0.8 mM MgSO₄, 5 mM glucose, pH7.4) and suspended at a ratio of 10 ml of the initial blood volume/mlbasic salt solution. Whole WBC population was used as a source of PMNs,with PMN numbers used per assay being adjusted according to the resultsof Wright's staining of the peripheral blood.

PMNs (2.5×10⁵/ml) were incubated with CAPE (0.05-5.0 nmol/ml) and/or TPA(25 pmol/ml) at 37° C. for 30 min in the presence of phenol red (100 μg)and HRPO (50 μg) (54). After the reaction was stopped with catalase (50μg/ml), the pH was adjusted to 12.5 with NaOH, the mixture wascentrifuged, and the absorbance of the supernatant was determined at 598nm. The concentration of H₂O₂ produced was established from the standardcurve.

B) Bovine Lens

Bovine lenses were obtained from a New Jersey slaughterhouse 3 h afterthe animals were sacrificed. Lenses were immediately immersed inartificial aqueous humor (AAH; 130 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 0.5 mMMgCl₂, 10 mM NaHCO₃, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (pH 7.3), and 2.5 mM D-glucose). Each of the bovine lenses in AAH(about 15 ml/lens) supplemented with 10 mM azide (a catalase inhibitor)was incubated with various amounts (0-1 μM) TPA at 37° C. for 24 h inthe presence or absence of CAPE (1 μM), as described for use of otherchemopreventive agents (31). For comparison, the lenses were alsoincubated with glucose (2.5 mM)/glucose oxidase (25 μM), aH₂O₂-generating system.

After incubation, lenses were homogenized in 15 ml of azide-supplementedAAH in a Waring Blender, and H₂O₂ content was determinedspectrofluorometrically, as described previously (55). Briefly, thehomogenates were incubated with 2′,7′-dichlorofluorescin diacetate,which is deacetylated by the esterase present in the homogenate. In thepresence of HRPO, the nonfluorescent substrate was oxidized by H₂O₂generated in the lens to a highly fluorescent product, which wasmeasured at excitation 475 nm and emission 525 nm (30, 31, 55). Theamount of H₂O₂ generated was determined from a standard curve, which wasconstructed by addition of increasing concentrations of H₂O₂ to ahomogenate derived from a control lens incubated only in AAH.

In some experiments, at the end of incubation [lenses in (a) AAH only(controls), (b) AAH+0.1 μM TPA, and (c) AAH+1 μM CAPE (30 min)+0.1 μMTPA] at 37° C. for 24 h, the lenses were photographed against a 3-mmgrid.

EXAMPLE 5

In Vivo Experiments on Rabbits

A) Chemicals and Reagents

12-O tetradecanoylphorbol-13 acetate (TPA) was purchased from LCServices Corp. (Woburn, Mass.). CAPE was synthesized as described,basically by acid-catalyzed esterification of caffeic acid withphenethyl alcohol (1).

B) Test Animals

Six healthy young (6-8 weeks old) white New Zealand rabbits of bothsexes were used for the experiments. Each animal had normal eyes asconfirmed by ocular examination by slit lamp.

C) Treatment of Animals and Monitoring the Eye Alterations

Cataracts were induced by the direct application of both TPA and CAPEinto the eye chamber. On every third day, 0.05 ml of the TPA solution(200 μg/ml PBS) was injected into the anterior chamber of both eyes viaa puncture at the corneo-scleral limbus with a fine 0.3×42 mm needlejoined to a tuberculin syringe. The animals were all given localanesthesia with HUMACAIN eye drops (human, Gödöllö, Hungary) understerile conditions for each application. One hour after TPA wasinjected, 0.05 ml of the CAPE solution (50 μg/ml) was administered intothe anterior chamber of only the left eye, as above. The right eye ofeach animal received no further treatment. This procedure was repeated atotal of 16 times. Periodic ocular examinations by slit lamp wereperformed after the 6th, 12th, and final drug applications.Subsequently, examinations were made on the 10th and 23rd days after thelast drug treatment. After each ocular examination, the physicalfindings were recorded and photodocumentation of the eye changes wereperformed. The photographic documentary was executed in the followingmanner. Firstly, lenses were illuminated by a fiber optic light sourcefrom behind and photographs were taken from a distance of 5-6 cm. Thediapositive black and white images on paper were transferred into an IBMpersonal computer (system 2, model 30) using a Sony-2-Cue CCD videocamera that was equipped with Fujinon TV zoom objective (1:1.8) via a17.5-105 signal-transfer system. Finally, with these computer imagereconstructions, the areas of the lens opacifications were delineatedand quantified using the Olympus image analyzer C-2 morphometry version2.2 program.

EXAMPLE 6

Mice were first treated with DMBA (7,12-dimethylbenz(a)anthracene) as aninitiating carcinogen, followed by tumor promotion with TPA(12-O-tetradecanoylphorbol-13-acetate). The effects of CAPE on DNA baseoxidation was studied by treating mice with CAPE during the promotion(treatment with TPA) phase. The results, which show that CAPE inhibitsoxidation of DNA bases, are as follows:

EFFECTS OF CAPE ON FORMATION OF HMdU IN DMBA/TPA-TREATED CD-1 MOUSE SKINDNA HMdU/ 10⁴ normal Group Treatment N bases (x ± SE) ↑^(b) or ↓^(c) P AAcetone/TPA 4 2.29 ± 1.97 — — B DMBA/TPA 9 12.58 ± 4.88  5.5x↑_(A);0.05⁺  2.3x↑_(G) C CAPE (1 nmol) 4 7.51 ± 2.49 40.3%↓ D CAPE (10 nmol) 40.92 ± 0.62 92.7%↓ 0.025^(*) E CAPE (100 nmol) 4 1.33 ± 1.00 89.4%↓0.025^(*) F CAPE (3000 nmol) 4 4.15 ± 0.96 67.0%↓ ^(d) G DMBA/Acetone 55.53 ± 1.38 2.4x↑_(A) ^(d) Legend ^(a)Groups C-F were initiated withDMBA and promoted with TPA in the presence of CAPE. ^(b)↑_(A): increasein comparison to the Acetone/TPA (Group A); ↑_(G): increase incomparison to the DMBA/Acetone (Group G). ^(c)↓: decrease in comparisonto the DMBA/TPA (Group B). N: number of mice separately analyzed.⁺significance versus Acetone/TPA (Group A). ^(*)significance versusDMBA/TPA (Group B). ^(d)0.1 ≧ p ≧ 0.05 versus DMBA/TPA (Group B).

EXAMPLE 7

CAPE was topically applied with 5 nmol of the tumor-promoter TPA to thebacks of DMBA-initiated female CD-1 mice twice weekly for 20 weeks.Papilloma formation was analyzed. Results are as follows:

Papilloma Inhibition Papilloma Volume CAPE (μmol) (per mouse) (permouse) 0.1 45% 53% 3 70% 74%

These results show that CAPE inhibits TPA-induced tumor promotion inmouse epidermis.

EXAMPLE 8

Cultured HeLa cells were treated with CAPE and the incorporation ofradiolabeled substrates into nucleic acids and protein was analyzed.Results are as follows:

Inhibition of Label Incorporation [³H] thymidine [³H] uridine [³H]leucine CAPE (μg/ml) into DNA into RNA into protein  5 32%  6% 0 10 75%18% 0 20 96% 54% 0

These data show that CAPE suppresses proliferation of cancer cells byinhibition of DNA and RNA synthesis.

RESULTS

CAPE-mediated Inhibition of TPA-induced PMN Infiltration, H₂O₂Production, and Formation of HMdUrd and 8-OHdGua in Mouse Skin. WhenSENCAR mice were treated with CAPE (6.5 nmol) 30 min prior to each oftwo TPA (6.5 nmol) applications, a number of inhibitory processesoccurred and are shown in FIGS. 2A-2B. Treatment of mice with CAPEdecreased TPA-induced PMN infiltration by ≈85%, as quantified by thepresence of MPO (FIG. 2A), an enzyme characteristic of PMNs (59).Although other cell types (i.e. monocytes) also contain MPO (59),treatment of mice was so short (21 h total), that it is unlikely thatcells other than PMNs would infiltrate the mouse skin in significantamounts during that time. Concurrently, topical application of CAPE alsoalmost completely (86%) inhibited TPA-induced H₂O₂ formation (FIG. 2B).HMdUrd levels were decreased by CAPE (FIG. 2C) below the acetone/acetonecontrols, while 8-OHdGua levels were suppressed significantly (70%)below controls (FIG. 2D).

The CAPE-mediated decrease of the levels of oxidized bases below theacetone/acetone controls indicated that the following have occurred: (a)acetone treatment evoked a weak inflammatory response; and/or (b) CAPEinhibited some normal oxidative processes in mouse skin as well. Toresolve this, additional controls were carried out. SENCAR mice, whichwere shaved 48 h before the experiment, were not treated with anything(blanks), and the MPO and 8-OHdGua levels were compared to mice treatedwith acetone. The 8-OHdGua level present in the epidermal DNA of theblanks was 65-70% lower than in the DNA of control acetone-treated mice.These results show that acetone treatment of control mice is alreadysomewhat inflammatory. Secondly, CAPE inhibits the inflammatoryprocesses down to the background levels represented by untreated blanks,particularly the oxidation of bases in DNA.

Infiltration of phagocytic cells and induction of edema have been usedas measures of tumor promoter-mediated inflammatory responses (3, 25,45, 46, 60). Therefore, it was puzzling why a low dose of 6.5 nmol CAPEsuppressed TPA-mediated PMN infiltration into mouse skin by 85% (FIG.2A), whereas over a 100-fold higher dose has been demonstrated asrequired to comparably inhibit ear edema in the same mouse strain (76).This could be because different sites were being analyzed, i.e. skin onthe back of the mouse versus ears. Another possible explanation would beif PMN infiltration was much more sensitive to the action ofchemopreventive agents than edema in general. To determine which of thetwo possibilities is more likely to be the contributing factor, bothears of SENCAR mice were simultaneously treated with CAPE/TPA. Earpunches from one ear were weighed to measure edema, and the other earwas used to determine MPO levels, a measure of PMN infiltration. FIG. 3shows the results of those paired experiments. It is apparent that underthe same treatment conditions, CAPE suppressed TPA-induced PMNinfiltration nearly 50% at 0.1 nmol of CAPE/ear, while a dose 4 ordersof magnitude higher (1 μmol) was needed for similar inhibition of edema.

Inhibitory Effect of CAPE on TPA-mediated H₂O₂ Production by Human PMNsin Vitro

It was shown previously that the levels of oxidized bases in DNA dependon the amounts of H₂O₂ generated (21, 22, 54). A decrease in the H₂O₂produced could be due to the decreased infiltration of PMNs. However, italso could be due to the CAPE-mediated inhibition of TPA-inducedoxidative burst of PMNs. To determine the latter, human PMNs werepretreated with various doses of CAPE (0.05-5.0 nmol CAPE/2.5×10⁵PMNs/ml) prior to TPA application (25 pmol TPA/2.5×10⁵ PMNs/ml). FIG. 4shows that similar to other chemopreventive agents, CAPE inhibited theoxidative burst of PMNs (as measured by H₂O₂ production) in adose-dependent manner, with 50% inhibition at 0.5 nmol CAPE/ml. Thismakes CAPE one of the most potent inhibitors of the oxidative burst thatwe have analyzed (28, 60-63). Since CAPE does not degrade H₂O₂ orinhibit HRPO (R. Bhimani and K. Frenkel, unpublished results), it isprobable that CAPE acts by inhibiting reactive oxygen species productionby TPA-activated PMNs.

Inhibitory Effect of CAPE on TPA-mediated Induction of H₂O₂ in BovineEye Lens

We decided to find out whether CAPE could also inhibit oxidative stress[known to contribute to cataract formation (39)] in the eye lens. First,we had to establish whether TPA can induce oxidant production in thelens. TPA is known to cause Ca²⁺ mobilization in a variety of cells(37-40), and Ca²⁺ was shown to play a role in the development ofcataracts (39, 68, 69).

The concentration of H₂O₂ was measured in lens homogenates aftertreatment of the whole lens with TPA or glucose/glucose oxidase, aH₂O₂-producing enzymatic system. As Table 1 shows, the 24-h incubationwith TPA resulted in increased formation of H₂O₂ in the bovine lens in adose-dependent manner, with 0.05, 0.1 and 1.0 μM TPA causing about 2-,3.5- and 7-fold increases in the concentration of H₂O₂ over that presentin control lenses in the absence of TPA. Table 1 also shows that 0.1 μMTPA-induced H₂O₂ was generated at comparable levels to those produced byglucose/glucose oxidase. Pretreatment of lenses with 1 μM CAPE for 30min followed by co-incubation with 0.1 μM TPA for 24 h caused a declinein TPA (0.1 μM)-induced H₂O₂ production by about 95% (Table 1).

TABLE 1 H₂O₂ formation in bovine lens incubated with either TPA,glucose/glucose oxidase and inhibitory effect of CAPE^(a) H₂O₂ % ofchange Treatment (μM) from control Control^(b) 11.9 ± 1.2Glucose/glucose oxidase 43.2 ± 1.3 365 0.01 μM TPA 11.3 ± 0.2  95 0.05μM TPA 23.3 ± 1.3 200 0.10 μM TPA 42.7 ± 1.6 360  1.0 μM TPA 83.8 ± 5.6705 1 μM CAPE/0.1 μM TPA 13.1 ± 1.3 110 ^(a)After treatment, lenses werehomogenized as described in “Materials and Methods,” and H₂O₂concentration was determined by formation of fluorescent# 2′,7′-dichlorofluorescein. The fluorescent intensity of whole lenshomogenate is proportional to H₂O₂ concentration, which was obtainedfrom a calibration curve. # The results are expressed as mean of 3experiments ± SE. ^(b)Lenses were incubated in artificial aqueous humoronly.

FIGS. 5A-5C show the results of experiments in which the whole lens wasincubated with AAH only (control, FIG. 5A), or with 0.1 μM TPA in theabsence (FIG. 5B) or presence of 1 μM CAPE (FIG. 5C) for 24 h. Alreadyat 0.1 μM, TPA caused opacity of the lens (FIG. 5B), which was even morepronounced when the lens was incubated with 1 μM TPA (31). At theconcentration used, CAPE almost completely prevented opacification ofthe lens.

Inhibitory Effect of CAPE on TPA-induced Cataracts In Vivo

Since the daily application of a TPA-containing drops into the eyes ofrabbits during a 6 week period failed to induce any visible alterationin the transparency of the lenses in any of the treated animals, TPA wasinjected directly into the anterior eye chambers. The pathologicaleffects of this approach were clearly manifested. Cataracts of varioussizes and types were evident by ocular examination in both eyes startingafter the 12th TPA injection. Progression of the cataract developmentoccurred during the phases of subsequent drug treatments and during the33 days after these treatments. Physical changes included an increasingsize of the cataracts, the deeper extension of the cataracts intonuclear portions of the lenses, and the maturation of some of thecataracts. Comparing the lens appearance of both eyes of each animal,the left eye that received CAPE was significantly clearer, having beenspared the extensive opacification induced by TPA exposure. In fact, itwas apparent by the 12th treatment period that CAPE injections to theleft eyes of animals produced a prophylactic effect on incipientcataracts.

The effects of the TPA and TPA plus CAPE injections appear in FIGS. 6and 7. FIG. 6 shows the TPA-treated right eye of rabbit No. 5 at theconclusion of the experiment. In the center of the lens, a rather largearched subcortical condensed immature but developing cataract isevident. It appears to be spreading toward the nuclear region. At the 8o'clock position, 3 small cortical punctiform opacities are present, andat the 11 o'clock position 2 subcortical condensed immature dot-likecataracts are visible. In contrast, FIG. 7 shows the CAPE-treated lefteye of the same animal. In the center of this eye, a smallcapsular-subcapsular immature cataract is evident. Additionally, at the7 o'clock position of the lens rim another subcapsular immature linearcataract is present as well as another very small circumscribedsubcapsular immature cataract situated nearby and above it.

Similar results were obtained in the other test animals. In theCAPE-treated eyes cataracts were visible, but they were generallysmaller and more immature. Specifically, the cataracts in theCAPE-treated eyes were all sharply delineated and mostly superficialcortical and subcapsular in nature. Conversely, the eyes that receiveTPA alone showed larger, more mature cataracts that had spread deeplyinto the nuclear region.

The differences in the cataract appearances for the two treatments wasquantitated by morphometrical computer analysis at the termination ofthe experiment. The area of the TPA-induced cataracts proved to be muchlarger (2.2-44.2 times) in the eye that did not receive CAPE than in theone injected with this compound. This result was obtained in 5 out ofthe 6 test animals and is shown in FIG. 8. In only a single rabbit wasthe cataract size in the CAPE-treated contralateral eye larger than thatnoted in the right one. By the Student's t-test, these differencesquantitated by morphometry were highly significant (p<0.001). Thequantitative basis for FIG. 8 is presented in Table 2. The cataractsizes are represented in relative units and the percent of CAPE-treatedcataracts are compared with the control right eye of each animal.

TABLE 2 Size of cataract in relative units. % of Animal Control¹ CAPE²Control 1 63.139 1.428 2.25 2 14.704 6.739 45.83 3 104.97 10.77 10.26 4111.62 28.722 25.73 5 99.923 30.199 30.22 6 28.578 43.184 151.11¹TPA-treated right eye. ²TPA + CAPE treated left eye of the same animal.

DISCUSSION

Propolis, which is a product of honeybee hives, has been used forcenturies in folk medicine as an anti-inflammatory agent (1). CAPE,which is an active ingredient present in propolis, is not toxic tonormal tissue. We have shown that CAPE is a potent inhibitor of a numberof oxidative processes both in vitro and in vivo even at very low doses(0.1-6.5 nmol/treatment). These processes include: (a) tumorpromoter-mediated PMN infiltration into mouse skin and mouse ears; (b)the TPA-induced oxidative burst by human PMNs and probably also by mousePMNs; (c) H₂O₂ production in TPA-treated mouse skin; and (d) formationof oxidized bases in epidermal DNA isolated from in vivo-treated mouseskin, as determined by the presence of HMdUrd and 8-OHdGua.

At higher doses, CAPE additionally inhibits the following TPA-mediatedprocesses: (a) TPA-induced ear edema in SENCAR mice when the amount ofCAPE is 1 μmol/ear and TPA is 0.4 nmol/ear; (b) TPA (5 nmol)-induced ODCin CD-1 mice is inhibited by 55% by 1 μmol CAPE (76), while it takes 10μmol CAPE to inhibit by 62% the TPA (4 nmol)-induced ODC in SENCAR mice;(c) topical application of CAPE (650 nmol/treatment; 2× treatment, 20 hapart) decreased TPA-mediated PMN infiltration and the formation ofHMdUrd and 8-OHdGua in SENCAR mice. However, this reduction was not aseffective as that caused by the lower (6.5 nmol) dose of CAPE, which wasequimolar to that of TPA (Table 3). Only H₂O₂ levels were furtherdecreased by the higher dose of CAPE. These results suggest thatchemopreventive agents may be very effective at low but not necessarilyat high doses. We have noted a similar phenomenon occurring with otherprotective agents such as EGCG (30). Apparently, higher doses of thesesubstances may interfere with other cellular processes and, in effect,counteract their own protective action.

TABLE 3 CAPE-mediated effects on TPA-induced infiltration of PMNs, H₂O₂production, and formation of oxidized DNA bases in skin of SENCARmice^(a) MPO H₂O₂ Treatment units/cm² nmol/cm² HMdUrd/10⁴ 8-OHdGua/10⁵skin (4)^(b) skin (5) bases (2) bases (2) Acetone  0.1 ± 0.03 12.8 ± 0.816.2 ± 7.7 5.0 ± 2.0 TPA 23.7 ± 7.6  31.5 ± 0.7 39.4 ± 6.4 11.8 ± 6.3 (6.5 nmol) TPA/CAPE 5.9 ± 0.3 15.7 ± 2.2 13.1 ± 0.6 1.4 ± 0.8 (6.5 nmol)TPA/CAPE 10.8 ± 2.0  11.6 ± 0.6  30.5 ± 10.5 9.4 ± 1.1 (650 nmol)^(a)Mice were treated as described in the legend to FIG. 2, except thatin addition to the group pretreated with 6.5 nmol CAPE, another groupwas pretreated with 650 nmol # CAPE prior to TPA applications. Resultsare expressed as mean values of 2-5 experiments ± SE. Note that HMdUrdis calculated per 10⁴, while 8-OHdGua is per 10⁵ normal bases.^(b)Numbers in parentheses, number of experiments with CAPE; values foracetone controls or TPA treatments are based on over 20 determinationseach.

The very small amounts of CAPE required for protection from oxidativedamage indicate that these agents probably act by interfering with theoxidative activation of the cells rather than by being antioxidants,which would require much higher doses needed for scavenging of thereactive oxygen species already produced. That this may be the case isshown by findings that TPA-treated HeLa cells, which were preincubatedwith low doses (5-25 nmol) of EGCG, contained lower levels of HMdUrd and8-OHdGua than cells treated only with TPA (30). Similar to what we foundfor CAPE, higher doses of EGCG (50 nmol) caused a less effectiveinhibition of TPA-induced oxidative processes.

As we noticed, relatively high doses of CAPE (1-10 μmol) were needed toinhibit 4-5 nmol or 0.4-0.5 nmol of TPA-mediated ODC or edema induction,respectively, in two strains of mice (“Results” Section, supra, and 76).In contrast, for inhibition of phagocytic infiltration, reactive oxygenspecies production and oxidative DNA damage in one of those strains(SENCAR), a very low does of CAPE (6.5 nmol)/treatment, 2× treatment, 20h apart; equimolar to that of TPA) was extremely effective (FIG. 2). PMNinfiltration declined by 85%, HMdUrd formation by 115%, and that of8-OHdGua by near 170%. Both oxidized base derivatives were lowered belowthe levels present in the acetone-only treated controls, particularly8-OHdGua, which declined 70% below the basal levels. We previously foundthat sarcophytol A (an anti-tumor-promoting agent effective in atwo-stage carcinogenesis model in mice) at a dose (6.5 nmol) equimolarto that of the tumor promoter (10), was also a potent inhibitor of TPA(6.5 nmol/treatment)-induced oxidative processes (i.e. PMN infiltration,reactive oxygen species generation, and DNA base oxidation) in SENCARmice (4). That inhibition occurred during the same two-dose treatmentthat was utilized in the current study, as well as during a typicaltumor promotion regimen induced by 3.2 nmol TPA applied twice a week for16 weeks. Hence, it appears that it is PMN infiltration, reactive oxygenspecies production, and oxidized DNA base formation that correlate withand might be necessary for tumor promotion, whereas ODC and edemainduction alone (without the former three processes) might not besufficient for tumor promotion. Other investigators, using differentexperimental designs, also noted dissociation of ODC induction from therequirements for tumor promotion (45, 70).

The other ex vivo system utilized by us also yielded very interestingand potentially important information. TPA-induced H₂O₂ production inbovine lenses occurred in a dose-dependent manner, and it caused lensopacification. CAPE suppressed formation of TPA-induced H₂O₂ formationand opacification of lenses at a concentration that was 10-fold higher(1 μM) than the TPA (0.1 μM) that induced them. It is suspected thathigh Ca²⁺ induces lens opacity and causes formation of cataracts (39).Interestingly, it has been found that H₂O₂ enhances the activities ofNa⁺/Ca⁺ as well as of Na⁺/Ca⁺ exchangers and increases opacity of thebovine lens (31, 68, 69). Hence, it is possible that the H₂O₂ producedin response to TPA causes elevation in Ca²⁺ within the lens and,consequently, causes opacity as well. It is encouraging that thechemopreventive agents which suppress oxidative activation of cells alsocan protect the lens from opacification. Patients with cataracts wereshown to have highly elevated levels of H₂O₂ (39, 71). Ca²⁺ is also morereadily transported into the aging lens, which is more prone to cataractdevelopment (72). By enhancing the activity of the Na⁺/H⁺ exchanger,H₂O₂ could cause more Na²⁺ accumulation within and less Na⁺ outside thelens, leading to a higher influx of Ca²⁺ as well as elevation ofintracellular pH (68, 69, 73). Similar processes occur during theoxidative burst of PMNs (14) whether caused by TPA or opsonizedparticulates, with its attendant acidification around the PMN membrane.Moreover, Ca²⁺ ionophore also can induce the oxidative burst andactivate the NADPH oxidase that is responsible for production ofreactive oxygen species (75) as well as cause hydroperoxide productionin mouse epidermis in vivo (67).

We also showed that TPA is a potent inducer of cataracts in the eyes oflive rabbits and that CAPE injections following the TPA treatmentssignificantly reduced the pathological effects of this phorbol ester.

Thus, two seemingly diverse biological systems may have certain types ofresponses in common. Tumor promoter-treated mouse skin responds with PMNinfiltration and H₂O₂ production. Similarly, a TPA-treated lensgenerates large amounts of H₂O₂₁, which contributes to cataractformation. Both involve change in Ca²⁺ homeostasis, elevation of whichcan be damaging in both systems. It is of considerable interest thatTPA-induced H₂O₂ formation as well as opacity of lenses respond to CAPE.

REFERENCES

1. Grunberger, D., Banerjee, R., Eisinger, K., Oltz, E. M., Efros, L.,Caldwell, M. Estevez, V., and Nakanishi K. Preferential cytotoxicity ontumor cells by caffeic acid phenethyl ester isolated from propolis.Experientia (Basel), 44: 230-232. 1988

2. Su, Z-Z., Grunberger, D., and Fisher, P. B. Suppression of adenovirustype 5 EIA-mediated transformation and expression of the transformedphenotype by caffeic acid phenethyl ester (CAPE), Mol. Carcinog., 4:231-242, 1991.

3. Frenkel, K. Carcinogen-mediated oxidant formation and oxidative DNAdamage. Phamacol. Ther., 53: 127-166, 1992.

4. Kensler, T. W., and Taffe, B. G. Free radicals in tumor promotion.Adv. Free Radical Biol. Med., 2: 347-387, 1986.

5. D'Onofrio, C., Maly, F. E., Fischer, H., and Maas, D. Differentialgeneration of chemiluminescence detectable oxygen radicals by normalpolymorphonuclear leukocytes challenged with sera from systemic lupuserythematosus and rheumatoid arthritis patients. Klin. Wochenschr., 62:710-716, 1984.

6. Evans, C. R., Omorphos, S. C., and Baysal, E. Sickle cell membranesand oxidative damage. Biochem. J., 237: 265-269, 1986.

7. Frenkel, K., Chrzan, K., Troll, W., Teebor, G. W., and Steinberg, J.J. Radiation-like modification of bases in DNA exposed to tumorpromoter-activated PMN leukocytes. Cancer Res., 46: 5533-5540, 1986.

8. Birnboim, H. C. Factors which affect DNA strand breakage in humanleukocytes exposed to tumor promoter phorbol myristate acetate. Can. J.Physiol. Pharmacol., 60: 1359-1366, 1982.

9. Cheng, K. C., Cahill, D. S., Kasai, H., Nishimura, S., and Loeb, L.A. 8-Hydroxydeoxyguanosine, an abundant form of oxidative DNA damagecauses G-T and A-T substitutions. J. Biol. Chem., 267: 166-172, 1992.

10. Yamamoto, F., Kasai, H., Bessho, T., Chung, M. H., Inoue, H.,Obtsuka, E., Hori, T. and Nishimura, S. Ubiquitous presence in mammaliancells of enzymatic activity specifically cleaving 8-hydroxyguaninecontaining DNA. Jpn. J. Cancer Res., 83: 351-357, 1992.

11. Higgins, S., Frenkel, K., Cummings, A., and Teebor, G. Definitivecharacterization of human thymine glycol-N-glycosylase activity.Biochemistry, 26: 1683-1688, 1987.

12. Hollstein, M. C., Brooks, P., Linn, S., and Ames, B.Hydroxymethyluracil DNA glycosylase in mammalian cells. Proc. Natl.Acad. Sci. USA, 81: 4003-4007, 1984.

13. Teebor, G. W., Boorstein, R. J. and Cadet, J. The repairability ofoxidative free radical-mediated damage to DNA: a review. Int. J. Radiat.Biol., 54: 131-150, 1988.

14. Breimer, L. H. Molecular mechanisms of oxygen radical carcinogenesisand mutagenesis. The role of DNA base damage. Mol. Carcinog., 3:188-197, 1990.

15. Tchou, J., and Grollman, A. P. Repair of DNA containing theoxidatively-damaged base, 8-oxoguanine. Mutat. Res., 299: 277-287, 1993.

16. Waschke, S., Reefschlager, J., Barwolff, D., and Langen, P.5-Hydroxymethyl-2′-deoxyuridine, a normal DNA constituent in certainBacillus subtilis phages is cytostatic for mammalian cells. Nature(Lond.), 225: 629-630, 1975.

17. Shirname-More, L., Rossman, T., Troll, W., Teebor, G., and Frenkel,K. Genetic effects of 5-hydroxymethyl-2′-deoxyuridine, a product ofionizing radiation. Mutat. Res., 178: 177-186, 1987.

18. Shigenaga, M. K., Gimeno, C-J., and Ames, B. K. Urinary8-hydroxy-2′-deoxyguanosine as a biological marker of in vivo oxidativeDNA damage. Proc. Natl. Acad. Sci. USA, 86: 9697-9701, 1989.

19. Floyd, R. A. The role of 8-hydroxyguanine in carcinogenesis.Carcinogenesis (Lond.). 11: 1447-1450, 1990.

20. Wei, H. and Frenkel, K. In vivo formation of oxidized bases in tumorpromoter-treated mouse skin. Cancer Res. 51: 4443-4449, 1991.

21. Wei, H. and Frenkel, K. Suppression of tumor promoter-inducedoxidative events and DNA damage in vivo by sarcophytol A: a possiblemechanism of antipromotion. Cancer Res. 52: 2298-2303, 1992.

22. Wei, H., and Frenkel K. Relationship of oxidative events and DNAoxidation in SENCAR mice to in vivo promoting activity of phorbolester-type tumor promoters. Carcinogenesis (London), 14: 1195-1201,1993.

23. Smart, R. C., Huang, M-T., Han, Z. T., Kaplan, M. C., Focella, A.,and Conney, A. H. Inhibition of 12-0-tetradecanoylphorbol-13-acetateinduction of ornithine decarboxylase, DNA synthesis, and tumor promotionin mouse skin by ascorbic acid and ascorbyl palmitate. Cancer Res., 47:6633-6638, 1987.

24. Huang, M. T., Smart, R. C., Wong, C-Q., and Conney, A. H. Inhibitoryeffect of curcumin, chlorogenic acid, caffeic acid, and ferulic acid ontumor promotion in mouse skin by 12-0-tetradecanoylphorbol-13-acetate.Cancer Res., 48: 5941-5946, 1988.

25. Huang, M-T., Lysz, T., Ferraro, T., Abidi, T. F., Laskin, J. D., andConney, A. H. Inhibitory effects of curcumin on in vitro lipoxygenaseand cyclooxygenase activities in mouse epidermis. Cancer Res., 52:813-819, 1991.

26. Agarwal, R., Katiyar, S. K., Zaidi, S.I.A., and Mukhtar, H.Inhibition of skin tumor promoter-caused induction of epidermalornithine decarboxylase in SENCAR mice by polyphenolic fraction isolatedfrom green tea and its individual epicatechin derivatives, Cancer Res.,52: 3582-3588, 1992.

27. Fujiki, H., H. Suganuma, M., Supuri, H., Yoshizawa, S., Takagi, K.,and Kobayashi, M. Sarcophytols A and B inhibit tumor promotion byteleocidin in two-stage carcinogenesis in mouse skin. J. Cancer Res.Clin. Oncol., 115: 25-28, 1989.

28. Frenkel, K., Zhong, Z., Rashid, K., and Fujiki, H. Sarcophytols andprotease inhibitors suppress H₂O₂ formation and oxidative DNA damage.In: O. F. Nygaard (ed.), Anticarcinogenesis and Radiation Protection,Vol. 2. pp 357-366. New York Plenum Publishing Corp., 1991.

29. Yoshizawa, S., Horiuchi, T., Fujiki, H., Yoshida, T., Okuda, T., andSugimura, T. Antitumor promoting activity of (−)epigallocatechingallate, the main constituent of “tannin” in green tea. Phytother. Res.,1: 44-47, 1987.

30. Bhimani, R., Troll, W., Grunberger, D., and Frenkel, K. Inhibitionof oxidative stress in HeLa cells by chemopreventive agents CancerResearch, 53: 4528-4533, 1993.

31. Ye, J., Frenkel, K., and Zadunaisky, J. A. Lens opacification andH₂O₂ elevation induced by a tumor promoter. Lens Eye Tox. Res., 9:37-48, 1992.

32. Bhuyan, K., Master, R. W. Coles, R. S., Bhuyan, D. K. Molecularmechanism of cataractogenesis: IV. Evidence of phospholipidmalondialdehyde adduct in human senile cataract. Mech. Ageing Dev. 34:289-296, 1986.

33. Varma, S. D., Chand, D., Sharma, Y. R., Kuck, J. F., Richards, R. D.Oxidative stress on lens and cataract formation: Role of light andoxygen. Curr. Eye Res. 3: 35-57, 1884.

34. Spector, A., Wang, G-M., Wang, R-R. Photochemically inducedcataracts in rat lenses can be prevented by AL-3823A, a glutathioneperoxidase mimic. Proc. Natl. Acad. Sci. USA. 90: 7485-7489, 1993.

35. Bhuyan, K. C., Bhuyan, D. K. Regulation of hydrogen peroxide in eyetumors. Effect of 3-amino-1H-1, 2, 4-triazole on catalase andglutathione peroxidase of rabbit eye. Biochim. Biophys. Acta. 497:641-651, 1977.

36. Bhuyan, K. C., Bhuyan, D. K. Superoxide dismutase of the eye:Relative functions of superoxide dismutase and catalase in protectingthe ocular lens from oxidative damage. Biochim. Biophys. Acta. 542:28-38, 1978.

37. Bhuyan, K. C., Bhuyan, D. K., Podos, S. M. Free radical enhancerxenobiotic as an inducer of cataract in rabbit. Free Radic. Res. Comm.12-13: 609-620, 1991.

38. Goosey, J. D., Zigler, J. S., Kinoshita, J. H. Cross linking of lenscrystallins in a photodynamic system: A process mediated by singletoxygen. Science 208: 1278-1280, 1980.

39. Spector, A. Aspects of the biochemistry of cataracts. The OcularLens, Structure, Function and Pathology. New York: Maisel, H, MarcelDekker Inc: 405-438, 1985.

40. Augusteyn, R. C. Protein modification in cataract: Possibleoxidative mechanism. Mechanism of Cataract Formation in the Human Lens.New York: Academic Press. 71-115, 1981.

41. Babizhayev, M. A. Accumulation of lipid proxidation products inhuman cataracts. Acta Ophthalmol. 67: 281-287, 1989.

42. Bhuyan, K. C., Bhuyan, D. K. Molecular mechanism ofcataractogenesis: III. Toxic metabolites of oxygen as initiators oflipid peroxidation and cataract Curr. Eye Res. 13: 67-81, 1984.

43. Bhuyan, K. C., Bhuyan, D. K., Podos, S. M. Lipid peroxidation incataract of the human. Life Sci. 38: 1463-1471, 1986.

44. Fischer, S. M., Baldwin, J. K., and Adams, L. M. Effects ofanti-promoters and strain of mouse on tumor promoter-induced oxidants inmurine epidermal cells. Carcinogenesis (Lond.), 7: 915-918, 1986.

45. Perchellet, E. M., and Perchellet, J-P. Characterization of thehydroperoxide response observed in mouse skin treated with tumorpromoter in vivo. Cancer Res., 49: 6193-6201, 1989.

46. Kensler, T. W., Egner, P. A. Taffe. B. G., and Trush, M. A. Role offree radicals in tumor promotion and progression. In: T. J. Slaga, A. J.P. Klein-Szanto, R. K. Boutwell, D. E. Stevenson, H. L. Spitzer, and B.D'Motto (eds). Skin Carcinogenesis Mechanisms and Human Relevance, pp.233-248. New York; Alan R. Liss. Inc., 1989.

47. Perchellet, J-P., and Perchellet, E. M. Antioxidants and multistagecarcinogenesis in mouse skin. Free Rad. Bio. Med., 7: 377-108, 1989.

48. Fesen, M. R., Kohn, K. W., Leteurtre, F., and Pommier, Y. Inhibitorsof Human Immunodeficiency Virus Integrase. Proc. Natl. Acad. Sci. USA.90: 2399-2403, 1993.

49. Kasai, H., and Nishimura, S. Hydroxylation of deoxyguanosine at theC-8 position by ascorbic acid and other reducing agents. Nucleic AcidsRes., 12: 2137-2145, 1984

50. Frenkel, K., Zhong, Z., Wei, H., Karkoszka, J., Patel, U., Rashid,K., Georgescu, M. and Solomon, J. Quantitative high-performance liquidchromatography analysis of DNA oxidized in vitro and in vivo. Anal.Biochem., 196: 126-136, 1991.

51. Matsuda, T. A., Shinozaki, M., Suzuki, M., Watanabe, K., andMiyasaka, T. Convenient method for the selective acylation of guaninenucleosides. Synthesis, 385-386, 1986.

52. Bradford, M. M. A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dyebinding. Anal. Biochem., 72: 248-254, 1976.

53. Metcalf. J. A., Gallin, J. I., Nauseef, W. M., and Root, R. K.Laboratory Manual of Neutrophil Function, pp. 150-151. New York: RavenPress, 1986.

54. Frenkel, K., and Chrzan, K. Hydrogen peroxide formation and DNA basemodification by tumor promoter-activated polymorphonuclear leukocytes.Carcinogenesis (Lond.). 8: 455-460, 1987.

55. Frenkel, K., and Gleichauf, C. Hydrogen peroxide formation by cellstreated with a tumor promoter. Free Rad. Res. Commun., 13: 783-794,1991.

56. Srimal, R. C., and Dhavan, B. N. Pharmacology of diferuloyl methane(curcumin), a non-steroidal anti-inflammatory agent. J. Pharm.Pharmacol., 25: 447-452, 1973.

57. Sharma, O. P. Antioxidant activity of curcumin and relatedcompounds. Biochem Pharmacol., 25: 1811-1812, 1976.

58. Rao, T. S., Basu, N., and Siddiqui, H. H. Anti-inflammatory activityof curcumin analogues, Indian J. Med. Res., 75: 574-578, 1982.

59. Warren, J. S., Johnson, K. J., and Ward, P. A. Oxygen radicals incell injury and cell death. Pathol. Immunopatho. Res. 6: 301-315, 1987.

60. Frenkel, K. Oxidation of DNA bases by tumor promoter-activatedprocesses. Environ Health Perspect. 81: 45-54, 1989.

61. Frenkel, K., Chrzan, K., Ryan, C. A. Wiesner, R., and Troll, W.Chymotrypsin specific protease inhibitors decrease H₂O₂ formation byactivated human polymorphonuclear leukocytes. Carcinogenesis (Lond.), 8:1207-1212, 1987.

62. Zhong, Z., Tius, M., Troll, W., Fujiki, H., and Frenkel, K.Inhibition of H₂O₂ formation by human polymorphonuclear leukocytes(PMNs) as a measure of anticarcinogenic activity Proc. Am. Assoc. CancerRes., 32: 127, 1991.

63. Lim, J. S., Frenkel, K., and Troll, W. Tamoxifen suppresses tumorpromoter-induced hydrogen peroxide formation by human neutrophils.Cancer Res., 52: 4969-4972, 1992.

64. Boynton, A. L., Whitfield, J. F., and Isaaks, R. J.Calcium-dependent stimulation of BALB/c 3T3 mouse cell DNA synthesis bya tumor-promoting phorbol ester (PMA). J. Cell. Physiol., 87: 25-32.1976.

65. Verma, A. K., and Boutwell, R. K. Intracellular calcium and skintumor promotion: Calcium regulation of the induction of epidermalornithine decarboxylase activity by tumor promoter12-O-tetradecanoylphorbol-13-acetate. Biochem. Biophys. Res. Commun.,101: 375-383, 1981.

66. Wirth, P. J., Yuspa, S. H., Thorgeirsson, S. S., and Hennings, H.Induction of common patterns of polypeptide synthesis andphosphorylation by calcium and 12-O-teradecanoylphorbol-13-acetate inmouse epidermal cell culture. Cancer Res., 47: 2831-2838. 1987.

67. Perchellet, E. M., Jones, D., and Perchellet, J-P. Ability of theCa²⁻ ionophores A23187 and tenomycin to mimic some of the effects of thetumor promoter 12-O-tetradecanoylphorbol-13-acetate on hydroperoxideproduction, ornithine decarboxylase activity, and DNA synthesis in mouseepidermis in vivo. Cancer Res., 50: 5806-5812, 1990.

68. Ye. J., and Zadunaisky, J. A. Study of the Ca²⁺/Na⁺ exchangemechanism in vesicles isolated from apical membranes of lens epitheliumof spiny dogfish (Sgualus acanthias) and bovine eye. Exp. Eye Res., 55:243-250, 1992.

69. Ye. J., and Zadunaisky, J. A. A Na⁺/H⁺ exchanger and its relation tooxidative effects of plasma membrane vesicles from lens fibers. Exp. EyeRes., 55: 251-260, 1992.

70. Fischer, S. M., Baldwin, J. K., Jasheway, D. W., Patrick, K. E., andCameron, G. S. Phorbol ester induction of 8-lipoxygenase in inbredSENCAR (SSIN) but not C57BL/6J mice correlated with hyperplasia, edema,and oxidant generation but not ornithine decarboxylase induction. CancerRes., 48: 658-664, 1988.

71. Spector, A., and Garner, W. H. Hydrogen peroxide and human cataract.Exp. Eye Res., 33: 673-681, 1981.

72. Duncan, G., Hightower, K. R., Gandolfi, S. A., Tomlinson, J., andMareini, G. Human lens membrane cation permeability increases with age.Invest. Ophthalmol. Vis. Sci. 30: 1855-1859, 1989.

73. Alvarez, J., Garcia-Sancho, J., Mollinedo, F. and Sanchez, A.Intracellular Ca²⁺ potentiates Na⁺/H⁺ exchange and cell differentiationinduced by phorbol ester in U937 cells. Eur. J. Biochem., 183: 709-714,1989.

74. Araki, A., Inoue, T., Cragoe, E. J., Jr., and Sendo, F. Na⁺/H⁺exchange modulates rat neutrophil mediated tumor cytotoxicity. CancerRes., 51: 3212-3216, 1991.

75. Follin, P., Johansson, A., and Dahlgren, C. Intracellular productionof reactive oxygen species in human neutrophils following activation bythe soluble stimuli FMLP, dioctanoylglycerol and ionomycin. CellBiochem. Funct., 9: 29-37, 1991.

76. Frenkel, K., Wei, H., Bhimani, R., Ye, J., Zadunaisky, J., Huang,M-T., Ferraro, T., Conney, A. H., and Grunberger, D. Inhibition of tumorpromoter-mediated processes in mouse skin and bovine lens by caffeicacid phenethyl ester. Cancer Research, 53: 1255-1261, 1993.

We claim:
 1. A method of preventing in a subject in need therof adisease resulting from oxidative stress wherein oxidative stress causesoxidation of DNA bases and the disease is selected from the groupconsisting of rheumatoid arthritis, lupus, sickle cell anemia andcancer, which comprises administering to the subject a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and aneffective oxidative stress-inhibiting amount of a compound having thestructure:

wherein X and Y are independently carbonyl, C═S, S═O, or O═S═O; n and pare independently 0 or 1; R¹ and R² are independently hydrogen, linearor branched C₁-C₁₈ alkyl, alkenyl, or alkynyl, either unsubstituted orsubstituted with halogen, including F, Cl, Br, and I, C₁-C₆ alkoxy,(C═O)R⁷, or (C═O)OR⁸; wherein R⁷ and R⁸ are independently C₁-C₆ linearor branched alkyl; R³, R⁴, and R⁵ are independently hydrogen, halogen,including F, Cl, Br, and I, trihalomethyl, C₁-C₁₈ linear or branchedalkyl, alkenyl, alkynyl, haloalkyl, alkoxy, alkoxyalkyl, alkylthio, or(C═O)R⁹, (C═O)OR¹⁰, O(C═O) R¹¹, (C═S)R¹², (C═S) OR¹³, O(C═S)R¹⁴,(S═O)R¹⁵, (S═O)OR¹⁶, or (O═S═O)OR¹⁷; wherein R¹⁹, R¹⁰, R¹¹, R¹², R¹³,R¹⁴, R¹⁵, R¹⁶, and R¹⁷ are independently C₁-C₁₈ linear or branchedalkyl, alkenyl, or alkynyl; and R⁶ is aryl or C₁-C₁₈ branched or linearalkyl, alkenyl, or alkynyl, either unsubstituted or substituted withhalogen, including F, Cl, Br, and I, C₁-C₆ alkoxy, (C═O)R¹⁸, (C═O)OR¹⁹,or aryl; wherein R¹⁸ and R¹⁹ are independently C₁-C₆ branched or linearalkyl; wherein when n and p are 0 and R1, R2, R3, R4, and R5 arehydrogen, R6 is not phenylethyl or a pharmaceutically acceptable saltthereof.
 2. The method of claim 1, wherein n and p are 0, and R¹, R₂,R₃, R₄, and R₅ are hydrogen.
 3. The method of claim 2, wherein R₆ ishexyl, butyl, ethyl, or phenylethyl.
 4. The method of claim 1, whereinthe subject is a mammal.
 5. The method of claim 4, wherein the subjectis a human.
 6. The method of claim 1, wherein the disease comprisestumor formation resulting from oxidative stress.
 7. A method ofinhibiting in a subject the progression of a disease resulting fromoxidative stress wherein oxidative stress causes oxidation of DNA basesand the disease is selected from the group consisting of rheumatoidarthritis, lupus, sickle cell anemia and cancer, which comprisesadministering to the subject a pharmaceutical composition comprising apharmaceutically acceptable carrier and an effective oxidativestress-inhibiting amount of a compound having the structure:

wherein X and Y are independently carbonyl, C═S, S═O, or O═S═O; n and pare independently 0 or 1; R¹ and R² are independently hydrogen, linearor branched C₁-C₁₈ alkyl, alkenyl, or alkynyl, either unsubstituted orsubstituted with halogen, including F, Cl, Br, and I, C₁-C₆ alkoxy,(C═O)R⁷, or (C═O)OR⁸; wherein R⁷ and R⁸ are independently C₁-C₆ linearor branched alkyl; R³, R⁴, and R⁵ are independently hydrogen, halogen,including F, Cl, Br, and I, trihalomethyl, C₁-C₁₈ linear or branchedalkyl, alkenyl, alkynyl, haloalkyl, alkoxy, alkoxyalkyl, alkylthio, or(C═O)R⁹, (C═O)OR¹⁰, O(C═O)R¹¹, (C═S)R¹², (C═S)OR¹³, O(C═S)R¹⁴, (S═O)R¹⁵,(S═O)OR¹⁶, or (O═S═O)OR¹⁷; wherein R¹⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵,R¹⁶, and RL¹⁷ are independently C₁-C₁₈ linear or branched alkyl,alkenyl, or alkynyl; R⁶ is aryl or C₁-C₁₈ branched or linear alkyl,alkenyl, or alkynyl, either unsubstituted or substituted with halogen,including F, Cl, Br, and I, C₁-C₆ alkoxy, (C═O)R¹⁸, (C═O)OR¹⁹, or aryl;wherein R¹⁸ and R¹⁹ are independently C₁-C₆ branched or linear alkyl;wherein when n and p are 0 and R1, R2, R3, R4, and R5 are hydrogen, R6is not phenylethyl or a pharmaceutically acceptable salt thereof.
 8. Themethod of claim 7, wherein n and p are 0, and R₁, R₂, R₃, R₄, and R₅ arehydrogen.
 9. The method of claim 8, wherein R₆ is hexyl, butyl, ethyl.10. The method of claim 7, wherein the subject is a mammal.
 11. Themethod of claim 7, wherein the subject is a human.
 12. The method ofclaim 7, wherein the disease comprises tumor formation resulting fromoxidative stress.